Formulations of suberoylanilide hydroxamic acid and methods for producing same

ABSTRACT

The present invention provides a pharmaceutical composition or crystalline composition with a specific dissolution profile, which comprises suberoylanilide hydroxamic acid or a pharmaceutically acceptable salt or hydrate thereof as an active ingredient. The present invention provides a process of producing said crystalline composition or pharmaceutical composition. The present invention also provides compositions with a specific particle size distribution.

FIELD OF THE INVENTION

The present invention provides a pharmaceutical composition orcrystalline composition with a specific dissolution profile, whichcomprises suberoylanilide hydroxamic acid or a pharmaceuticallyacceptable salt or hydrate thereof as an active ingredient. The presentinvention provides a process of producing said crystalline compositionor pharmaceutical composition. The present invention also providescompositions with a specific particle size distribution.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced byarabic numerals within parentheses. Full citations for thesepublications may be found at the end of the specification immediatelypreceding the claims.

Cancer is a disorder in which a population of cells has become, invarying degrees, unresponsive to the control mechanisms that normallygovern proliferation and differentiation. For many years there have beentwo principal strategies for chemotherapeutic treatment of cancer: a)blocking hormone-dependent tumor cell proliferation by interference withthe production or peripheral action of sex hormones; and b) killingcancer cells directly by exposing them to cytotoxic substances, whichinjure both neoplastic and normal cell populations.

Cancer therapy is also being attempted by the induction of terminaldifferentiation of the neoplastic cells (1). In cell culture modelsdifferentiation has been reported by exposure of cells to a variety ofstimuli, including: cyclic AMP and retinoic acid (2,3), aclarubicin andother anthracyclines (4).

Despite many advances in the field of oncology, the majority of solidtumors remain incurable in the advanced stages. Cytotoxic therapy isused in most cases, however, it often causes significant morbidity tothe patient without significant clinical benefit. Less toxic and morespecific agents to treat and control advanced malignancies are beingexplored.

There is abundant evidence that neoplastic transformation does notnecessarily destroy the potential of cancer cells to differentiate(1,5,6). There are many examples of tumor cells which do not respond tothe normal regulators of proliferation and appear to be blocked in theexpression of their differentiation program, and yet can be induced todifferentiate and cease replicating. A variety of agents, including somerelatively simple polar compounds (5,7-9), derivatives of vitamin D andretinoic acid (10-12), steroid hormones (13), growth factors (6,14),proteases (15,16), tumor promoters (17,18), and inhibitors of DNA or RNAsynthesis (4,19-24), can induce various transformed cell lines andprimary human tumor explants to express more differentiatedcharacteristics.

Histone deacetylase inhibitors such as suberoylanilide hydroxamide acid(SAHA), belong to this class of agents that have the ability to inducetumor cell growth arrest, differentiation and/or apoptosis (25). Thesecompounds are targeted towards mechanisms inherent to the ability of aneoplastic cell to become malignant, as they do not appear to havetoxicity in doses effective for inhibition of tumor growth in animals(26). There are several lines of evidence that histone acetylation anddeacetylation are mechanisms by which transcriptional regulation in acell is achieved (27). These effects are thought to occur throughchanges in the structure of chromatin by altering the affinity ofhistone proteins for coiled DNA in the nucleosome. There are five typesof histones that have been identified in nucleosomes (designated H1,H2A, H2B, H3 and H4). Each nucleosome contains two of each histone typewithin its core, except for H1, which is present singly in the outerportion of the nucleosome structure. It is believed that when thehistone proteins are hypoacetylated, there is a greater affinity of thehistone to the DNA phosphate backbone. This affinity causes DNA to betightly bound to the histone and renders the DNA inaccessible totranscriptional regulatory elements and machinery. The regulation ofacetylated states occurs through the balance of activity between twoenzyme complexes, histone acetyl transferase (HAT) and histonedeacetylase (HDAC). The hypoacetylated state is thought to inhibittranscription of associated DNA. This hypoacetylated state is catalyzedby large multiprotein complexes that include HDAC enzymes. Inparticular, HDACs have been shown to catalyze the removal of acetylgroups from the chromatin core histones.

SAHA (ZOLINZA™ (vorinostat)) has been shown to be useful for treatingcancer, selectively inducing terminal differentiation of neoplasticcells, inducing cell growth arrest and/or inducing apoptosis. Theinhibition of HDAC by SAHA is thought occur through direct interactionwith the catalytic site of the enzyme as demonstrated by X-raycrystallography studies (28). The result of HDAC inhibition is notbelieved to have a generalized effect on the genome, but rather, onlyaffects a small subset of the genome (29). Evidence provided by DNAmicroarrays using malignant cell lines cultured with a HDAC inhibitorshows that there are a finite (1-2%) number of genes whose products arealtered. For example, cells treated in culture with HDAC inhibitors showa consistent induction of the cyclin-dependent kinase inhibitor p21(30). This protein plays an important role in cell cycle arrest. HDACinhibitors are thought to increase the rate of transcription of p21 bypropagating the hyperacetylated state of histones in the region of thep21 gene, thereby making the gene accessible to transcriptionalmachinery. Genes whose expression is not affected by HDAC inhibitors donot display changes in the acetylation of regional associated histones(31).

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition with aspecific dissolution profile, which comprises suberoylanilide hydroxamicacid or a pharmaceutically acceptable salt or hydrate thereof as anactive ingredient. In one embodiment, the active ingredient of thepharmaceutical composition has an in vitro dissolution profile with asimilarity factor (f2) of at least 50 to 100 compared to the referencedissolution profile shown in FIG. 1. The invention also providespharmaceutical compositions for oral administration, and unit dosageforms based thereon.

The present invention also provides a crystalline composition comprisingsuberoylanilide hydroxamic acid or a pharmaceutically acceptable salt orhydrate thereof as an active ingredient, wherein about 100 mg of theactive ingredient has an in vitro dissolution profile with a similarityfactor (f2) of at least 50 to 100 compared to the reference dissolutionprofile shown in FIG. 2.

The present invention also provides methods of producing thepharmaceutical compositions. The invention also provides compositionswith specific particle size distributions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the dissolution profile of SAHA from the reference capsulelot 0683_(—)004A001. The capsules contain about 100 mg of activeingredient SAHA, and excipients.

FIG. 2 shows the dissolution profile of the reference SAHA API batch1007D (blended SAHA crystals) prior to encapsulation. The dissolutionprofile was measured based on about 100 mg of SAHA.

FIG. 3 shows the particle size distribution of the capsule content ofpharmaceutical capsules of the invention. The capsules contain about 100mg of active ingredient SAHA, and excipients.

FIG. 4 shows the particle size distribution of the active ingredientSAHA from different batches prior to encapsulation (API).

FIG. 5 shows the dissolution profiles of SAHA from pharmaceuticalcapsules of the invention. The capsules contain about 100 mg of activeingredient SAHA, and excipients.

FIG. 6 shows the dissolution profiles of SAHA API batches (blended SAHAcrystals) prior to encapsulation. The dissolution profiles were measuredbased on about 100 mg of SAHA.

FIG. 7 shows x-ray diffractograms for SAHA. FIG. 7A-E: SAHA Form I-V.

FIG. 8 shows the dissolution profiles predicted by the computer model(curve) and the experimental dissolution profiles (indicated by dots,triangles and squares) for the reference sample (target), capsules 288and 283.

FIG. 9 shows the f2 values in relation to the fraction of API 288 in ablend with API 283 for different capsule densities.

FIG. 10 shows the impact of encapsulation conditions on the SAHAdissolution in capsules made from the blending containing 30% wet-milledAPI 288 and 70% unmilled API 283.

FIG. 11 shows the correlation between breakage rate constant and densityof capsule content.

FIG. 12 shows the normalized particle size distribution of ActiveIngredient (API) from different batches of SAHA capsules.

FIG. 13 shows the particle size distribution of capsule content from LotC0666001.

FIG. 14 shows the particle size distribution of capsule content from LotC0667001.

FIG. 15 shows mean serum concentrations of vorinostat followingadministration of a single oral dose in the fasted state and following ahigh-fat meal.

FIG. 16 shows mean serum concentrations of vorinostat followingadministration of 400 mg single or multiple oral doses following ahigh-fat meal.

DETAILED DESCRIPTION OF THE INVENTION

The term “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration, which wouldmaintain the specified dissolution rate of the active ingredient in thepharmaceutical composition. Suitable carriers are described in the mostrecent edition of Remington's Pharmaceutical Sciences, a standardreference text in the field, which is incorporated herein by reference.Liposomes and non-aqueous vehicles such as fixed oils may also be used.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the active ingredient, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

The term “f2” or “F2” refers to a similarity factor determined through apoint by point comparison of a new in vitro dissolution profile to areference in vitro dissolution profile, as shown in equation 1.

$\begin{matrix}{f_{2} = {50\log \left\{ {\left\lbrack {1 + {{1/n}{\sum\limits_{t = 1}^{n}\left( {R_{t} - T_{t}} \right)^{2}}}} \right\rbrack^{- 0.5} \times 100} \right\}}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

R_(t) refers to the percent of compound dissolved at each time point (t)for the reference. T_(t) refers to the percent of compound dissolved ateach time point (t) for the test sample. n refers to the number of timepoints used for the calculation. f₂ values of 50 or greater areconsidered to reflect similar in vitro dissolution rates.

For the purpose of this invention, dissolution rates or profiles invitro of the entire active ingredient of the pharmaceutical compositionis measured from the entire pharmaceutical composition according to thesteps and conditions in Example 14. In one embodiment, dissolution ratesor profiles in vitro is measured by using a USP Dissolution Apparatus IIwith a helical sinker (Quality Lab Accessories L.L.C., Manville, N.J.)in 900 mL of 2.0% Tween (TCI America, Portland, Oreg.) at a temperatureof 37±0.5° C., and paddles rotated at 100 rpm. The entire pharmaceuticalcomposition includes the entire active ingredient and if thepharmaceutical composition contains a capsule shell, carrier, excipient,diluent, disintegrating agent, lubricant, binder or any additional agentdescribed in the Pharmaceutical Composition Section below, themeasurement is performed with those components.

For the purpose of this invention, dissolution rates or profiles invitro of “a portion of the single oral dosage unit form comprising about100 mg of the active ingredient” is measured by retrieving a compositioncomprising about 100 mg of the active ingredient from the single oraldosage unit form, and using a USP Dissolution Apparatus II with ahelical sinker (Quality Lab Accessories L.L.C., Manville, N.J.) in 900mL of 2.0% Tween (TCI America, Portland, Oregon) at a temperature of37±0.5° C., and paddles rotated at 100 rpm. If the single oral dosageunit form contains a capsule shell, carrier, excipient, diluent,disintegrating agent, lubricant, binder or any additional agentdescribed in the Pharmaceutical Composition Section below, themeasurement is performed with those components.

Dissolution rates or profiles in vitro of “about 100 mg of the activeingredient of the pharmaceutical composition” is measured according tothe steps and conditions in Example 15. In one embodiment, it ismeasured by using a USP Dissolution Apparatus II with a helical sinker(Quality Lab Accessories L.L.C., Manville, N.J.) in 900 mL of 2.0% Tween(TCI America, Portland, Oreg.) at a temperature of 37±0.5° C., andpaddles rotated at 100 rpm.

For the purpose of this invention, particle size distribution (% volumeat each particle size) is measured via a Sympatec laser diffractionanalyzer (HELOS H1006, Clausthal-Zellerfeld, Germany) equipped with aRODOS powder dispersion system. The sample is atomized through a laserbeam using 0.1 bar air pressure, and particle size distribution iscollected using a focal length lens of 850 or 1750-μm with targetedobscuration range of 5-20%. A fraunhofer optical model is utilized todeconvolute the sample scattering patterns to yield the resultantparticle size distributions.

For the purpose of this invention, % volume of active ingredient ismeasured by retrieving the particle content (i.e., active ingredient andthe excipients) from the pharmaceutical composition, measuring theparticle size distribution (% volume of each particle size) of theparticle content, subtracting the particle size distribution ofparticles that are not active ingredient, and normalizing % volume ofactive ingredient. The % volume of active ingredient is normalized bymultiplying the % volume by 100%/percentage of active ingredientrelative to particle content.

The term “about” when used in the context of an amount refers to ±10% ofthe specified amount.

For the purpose of this invention, for X-ray diffraction patterns,depending on the calibration, sample or instrumentation, peaks at 2θ canshift up to ±0.3 degrees (error). In one embodiment, all peaks in X-raydiffraction pattern shift up to +0.3 degrees, or up to −0.3 degrees. AnX-ray diffraction pattern or peaks within that error is considered thesame or substantially similar.

Compositions with Specific Dissolution Rate

The present invention provides a pharmaceutical composition comprisingsuberoylanilide hydroxamic acid or a pharmaceutically acceptable salt orhydrate thereof as an active ingredient, wherein the entire activeingredient of the pharmaceutical composition is 43-63% dissolved at 10minutes, 66-86% dissolved at 30 minutes, and 77-97% dissolved at 60minutes in vitro. In one embodiment, the entire active ingredient of thepharmaceutical composition is 52-72% dissolved at 15 minutes, 66-86%dissolved at 30 minutes, and 73-93% dissolved at 45 minutes in vitro. Inanother embodiment, the entire active ingredient of the pharmaceuticalcomposition is 43-63% dissolved at 10 minutes, 52-72% dissolved at 15minutes, 58-78% dissolved at 20 minutes, 66-86% dissolved at 30 minutes,73-93% dissolved at 45 minutes and 77-97% dissolved at 60 minutes invitro. In one embodiment, the entire active ingredient of thepharmaceutical composition is 46-60% dissolved at 10 minutes, 55-69%dissolved at 15 minutes, 61-75% dissolved at 20 minutes, 69-83%dissolved at 30 minutes, 76-90% dissolved at 45 minutes, and 80-94%dissolved at 60 minutes in vitro. In one embodiment, at least 45% butless than or equal to 75% of the entire active ingredient is dissolvedat 15 minutes, at least 75% of the entire active ingredient is dissolvedin 60 minutes.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising suberoylanilide hydroxamic acid or apharmaceutically acceptable salt or hydrate thereof as an activeingredient, wherein the entire active ingredient of the pharmaceuticalcomposition has an in vitro dissolution profile with a similarity factor(f2) of at least 50 to 100 compared to the reference dissolution profileshown in FIG. 1. In one embodiment, f2 is 56 to 100. In one embodiment,f2 is 60 to 100. In one embodiment, f2 is 65 to 100. In anotherembodiment, f2 is 80 to 100.

In one embodiment, the active ingredient is crystalline. In anotherembodiment, the active ingredient is crystalline suberoylanilidehydroxamic acid. In a particular embodiment, the crystallinesuberoylanilide hydroxamic acid is SAHA Form I and characterized by anX-ray diffraction pattern substantially similar to that set forth inFIG. 7A. In one embodiment, crystalline suberoylanilide hydroxamic acidis characterized by an X-ray diffraction pattern includingcharacteristic peaks at 9.0, 9.4, 17.5, 19.4, 20.0, 24.0, 24.4, 24.8,25.0, 28.0 degrees 2θ.

In one embodiment, SAHA Form I is characterized by an X-ray diffractionpattern including characteristic peaks at about 9.0, 9.4, 17.5, 19.4,20.0, 24.0, 24.4, 24.8, 25.0, 28.0, and 43.3 degrees 2θ. In oneembodiment, crystalline suberoylanilide hydroxamic acid is characterizedby an X-ray diffraction pattern including characteristic peaks at 9.0,9.4, 17.5, 19.4, 20.0, 24.0, 24.4, 24.8, 25.0, 28.0, 43.3 degrees 2θ andlacking a peak at 13.4-14.0 and 22.7-23.0 degrees 2θ. In one embodiment,crystalline suberoylanilide hydroxamic acid is characterized by an X-raydiffraction pattern including characteristic peaks at 9.0, 9.4, 17.5,19.4, 20.0, 24.0, 24.4, 24.8, 25.0, 28.0 degrees 2θ and lacking a peakat 13.4-14.0 and 22.7-23.0 degrees 2θ. In one embodiment, SAHA Form I isadditionally characterized by the lack of at least one peak at about<8.7, 10.0-10.2, 13.4-14.0, 15.0-15.2, 17.5-19.0, 20.1-20.3, 21.1-21.3,22.0.-22.22, 22.7-23.0, 25.0-25.5, 26.0-26.2, and 27.4-27.6 degrees 2θ.In another embodiment, SAHA Form I is further characterized by aDifferential Scanning Calorimetry (DSC) thermogram having a singlemaximum value at about 164.4±2.0, as measured by a Perkins Elmer DSC 6Instrument. 5. In one embodiment, the crystalline suberoylanilidehydroxamic acid has unit cell parameters of a=10.9 Å, b=7.9 Å, c=16.4 Å,α=90°, β=97.8°, γ=90°, space group P2₁/n.

In a particular embodiment, the crystalline suberoylanilide hydroxamicacid is SAHA Form IV and is characterized by an X-ray diffractionpattern including characteristic peaks at about 8.8, 9.3, 11.0, 12.4,17.4, 19.4, 19.9, 22.4, 22.9, 23.83, 24.2, 24.8, 25.8, 27.0, 27.8, 28.4degrees 2θ.

In one embodiment, the invention provides a single capsule comprisingabout 100 mg suberoylanilide hydroxamic acid or a pharmaceuticallyacceptable salt or hydrate thereof as an active ingredient, wherein theentire active ingredient has an in vitro dissolution profilecharacterized by: at least 45% but less than or equal to 75% of theentire active ingredient is dissolved at 15 minutes, at least 75% of theentire active ingredient is dissolved in 60 minutes, wherein the activeingredient is crystalline suberoylanilide hydroxamic acid andcharacterized by an X-ray diffraction pattern including characteristicpeaks at 9.0, 9.4, 17.5, 19.4, 20.0, 24.0, 24.4, 24.8, 25.0, 28.0degrees θ, and lacking a peak at 13.4-14.0 and 22.7-23.0 degrees 2θ.

In another embodiment, the invention provides a single capsulecomprising about 100 mg suberoylanilide hydroxamic acid or apharmaceutically acceptable salt or hydrate thereof as an activeingredient, wherein the entire active ingredient has an in vitrodissolution profile with a similarity factor (f2) of at least 50 to 100compared to the reference dissolution profile shown in FIG. 1, whereinthe active ingredient is crystalline suberoylanilide hydroxamic acid andcharacterized by an X-ray diffraction pattern including characteristicpeaks at 9.0, 9.4, 17.5, 19.4, 20.0, 24.0, 24.4, 24.8, 25.0, 28.0degrees 2θ, and lacking a peak at 13.4-14.0 and 22.7-23.0 degrees 2θ.

In a further embodiment, the invention provides a single capsulecomprising about 100 mg suberoylanilide hydroxamic acid or apharmaceutically acceptable salt or hydrate thereof as an activeingredient, wherein the entire active ingredient has an in vitrodissolution profile characterized by 43-63% dissolved at 10 minutes,66-86% dissolved at 30 minutes, and 77-97% dissolved at 60 minutes,wherein the active ingredient is crystalline suberoylanilide hydroxamicacid and characterized by an X-ray diffraction pattern includingcharacteristic peaks at 9.0, 9.4, 17.5, 19.4, 20.0, 24.0, 24.4, 24.8,25.0, 28.0 degrees 2θ, and lacking a peak at 13.4-14.0 and 22.7-23.0degrees 2θ.

The invention also provides a single oral dosage unit form comprisingabout 120 mg to about 600 mg of suberoylanilide hydroxamic acid or apharmaceutically acceptable salt or hydrate thereof as an activeingredient, wherein a portion of said dosage unit form comprising about100 mg of the active ingredient has an in vitro dissolution profile witha similarity factor (f2) of at least 50 to 100 compared to the referencedissolution profile shown in FIG. 1. In one embodiment, the in vitrodissolution profile has a similarity factor (f2) of at least 70 to 100compared to the reference dissolution profile shown in FIG. 1. In oneembodiment, the active ingredient is crystalline suberoylanilidehydroxamic acid and characterized by an X-ray diffraction patternincluding characteristic peaks at 9.0, 9.4, 17.5, 19.4, 20.0, 24.0,24.4, 24.8, 25.0, 28.0 degrees 2θ, and lacking a peak at 13.4-14.0 and22.7-23.0 degrees 2θ.

The present invention also provides a crystalline composition comprisingsuberoylanilide hydroxamic acid or a pharmaceutically acceptable salt orhydrate thereof as an active ingredient, wherein about 100 mg of theactive ingredient has an in vitro dissolution profile with a similarityfactor (f2) of at least 50 to 100 compared to the reference dissolutionprofile shown in FIG. 2. This crystalline composition is a precursor tothe pharmaceutical composition. In the instance where the pharmaceuticalcomposition is in the form of a capsule, the crystalline composition isthe active ingredient with or without excipients before encapsulation.In one embodiment, the active ingredient is crystalline suberoylanilidehydroxamic acid and characterized by an X-ray diffraction patternincluding characteristic peaks at 9.0, 9.4, 17.5, 19.4, 20.0, 24.0,24.4, 24.8, 25.0, 28.0 degrees 2θ, and lacking a peak at 13.4-14.0 and22.7-23.0 degrees 2θ.

The active ingredient can be in any crystalline form provided that theactive ingredient particles exhibit the specified dissolution rate. Theactive ingredient can also be in amorphous form. The active ingredientparticles may be micronized, or may be agglomerated, particulategranules, powders, oils, oily suspensions or any other form of solid.

In a particular embodiment of the above compositions, the activeingredient is suberoylanilide hydroxamic acid.

The invention also encompasses pharmaceutical compositions comprisingpharmaceutically acceptable salts of the SAHA with inorganic bases, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The invention also encompasses pharmaceutical compositions comprisinghydrates of SAHA. The term “hydrate” includes but is not limited tohemihydrate, monohydrate, dihydrate, trihydrate and the like.

Compositions with Specific Particle Size Distribution

The invention also provides a pharmaceutical composition comprisingsuberoylanilide hydroxamic acid or a pharmaceutically acceptable salt orhydrate thereof as an active ingredient, wherein the % volume forparticle sizes from about 90 to 110 microns to about 120 to 250 micronsincreases, peaks at about 120 to 250 microns, and decreases after thepeak. In one embodiment, the peak is the highest % volume compared to %volume of other particle sizes.

In one embodiment, the % volume of active ingredient with particle sizeat about 90 to 110 microns is in the range of about 2.0% to about 10%,and the % volume of active ingredient with particle size at about 120 to250 microns is in the range of about 4.0% to about 12%. In oneembodiment, the % volume of active ingredient with particle size atabout 90 to 110 microns is in the range of about 3.0% to about 9%, andthe % volume of active ingredient with particle size at about 120 to 250microns is in the range of about 5.0% to about 11.5%.

In another embodiment, the % volume of particles with particle size atabout 90 to 110 microns is in the range of about 5.5% to about 8.0%, andthe % volume of particles with particle size at about 120 to 250 micronsis in the range of about 6.5% to about 9.0%. In one embodiment, the %volume of particles with particle size at about 90 to 110 microns is inthe range of about 6.0% to about 7.5%, and the % volume of particleswith particle size at about 120 to 250 microns is in the range of about7.0% to about 8.5%.

In one embodiment, the % volume of active ingredient with particle sizeless than about 105 microns is about 45-85% and the % volume of activeingredient with particle size more than about 105 microns is about55-15%.

In one embodiment, the % volume of active ingredient for particle sizesfrom about 20 to 25 microns to about 35 to 40 microns increases, peaksat about 35 to 40 microns, and decreases after the peak. In oneembodiment, the % volume of active ingredient with particle size atabout 20 to 25 microns is in the range of about 1.0% to about 4%, andthe % volume of active ingredient with particle size at about 35 to 40microns is in the range of about 3.0% to about 7%.

In one embodiment, the active ingredient is crystalline. In anotherembodiment, the active ingredient is crystalline suberoylanilidehydroxamic acid. In a particular embodiment, the crystallinesuberoylanilide hydroxamic acid is SAHA Form I and characterized by anX-ray diffraction pattern substantially similar to that set forth inFIG. 7A. In one embodiment, crystalline suberoylanilide hydroxamic acidis characterized by an X-ray diffraction pattern includingcharacteristic peaks at 9.0, 9.4, 17.5, 19.4, 20.0, 24.0, 24.4, 24.8,25.0, 28.0 degrees 2θ.

In one embodiment, SAHA Form I is characterized by an X-ray diffractionpattern including characteristic peaks at about 9.0, 9.4, 17.5, 19.4,20.0, 24.0, 24.4, 24.8, 25.0, 28.0, and 43.3 degrees 2θ. In oneembodiment, crystalline suberoylanilide hydroxamic acid is characterizedby an X-ray diffraction pattern including characteristic peaks at 9.0,9.4, 17.5, 19.4, 20.0, 24.0, 24.4, 24.8, 25.0, 28.0, 43.3 degrees 2θ andlacking a peak at 13.4-14.0 and 22.7-23.0 degrees 2θ. In one embodiment,crystalline suberoylanilide hydroxamic acid is characterized by an X-raydiffraction pattern including characteristic peaks at 9.0, 9.4, 17.5,19.4, 20.0, 24.0, 24.4, 24.8, 25.0, 28.0 degrees 2θ and lacking a peakat 13.4-14.0 and 22.7-23.0 degrees 2θ. In one embodiment, SAHA Form I isadditionally characterized by the lack of at least one peak at about<8.7, 10.0-10.2, 13.4-14.0, 15.0-15.2, 17.5-19.0, 20.1-20.3, 21.1-21.3,22.0.-22.22, 22.7-23.0, 25.0-25.5, 26.0-26.2, and 27.4-27.6 degrees 2θ.In another embodiment, SAHA Form I is further characterized by aDifferential Scanning Calorimetry (DSC) thermogram having a singlemaximum value at about 164.4±2.0, as measured by a Perkins Elmer DSC 6Instrument. 5. In one embodiment, the crystalline suberoylanilidehydroxamic acid has unit cell parameters of a=10.9 Å, b=7.9 Å, c=16.4 Å,α=90°, β=97.8°, γ=90°, space group P2₁/n.

In a particular embodiment, the crystalline suberoylanilide hydroxamicacid is SAHA Form IV and is characterized by an X-ray diffractionpattern including characteristic peaks at about 8.8, 9.3, 11.0, 12.4,17.4, 19.4, 19.9, 22.4, 22.9, 23.83, 24.2, 24.8, 25.8, 27.0, 27.8, 28.4degrees 2θ.

Pharmaceutical Compositions

The active ingredient can be incorporated into pharmaceuticalcompositions suitable for oral administration. The active ingredient mayoptionally be incorporated with a pharmaceutically acceptable carrier orexcipient. In one embodiment, the pharmaceutically accetaptable carrieris in solid particle form. Any inert excipient that is commonly used asa carrier or diluent may be used in the formulations of the presentinvention, such as for example, a gum, a starch, a sugar, a cellulosicmaterial, an acrylate, or mixtures thereof. In one embodiment, thediluent is microcrystalline cellulose. The compositions may furthercomprise a disintegrating agent (e.g., croscarmellose sodium) and alubricant (e.g., magnesium stearate), and in addition may comprise oneor more additives selected from a binder, a buffer, a proteaseinhibitor, a surfactant, a solubilizing agent, a plasticizer, anemulsifier, a stabilizing agent, a viscosity increasing agent, asweetener, a film forming agent, or any combination thereof.Furthermore, the compositions of the present invention may be in theform of controlled release or immediate release formulations.

In one embodiment, the pharmaceutical composition described herein mayfurther be comprised of microcrystalline cellulose, croscarmellosesodium and magnesium stearate. The percentage of the active ingredientand various excipients in the formulations may vary. For example, thecomposition may comprise between about 20 and 90%, between about 50-80%or between about 60-70% by weight of the active ingredient. Furthermore,the composition may comprise between about about 10 and 70%, betweenabout 20-40%, between about 25-35% by weight microcrystalline celluloseas a carrier or diluent. Furthermore, the composition may comprisebetween about 1 and 30%, between about 1-10%, between about 2-5% byweight croscarmellose sodium as a disintegrant. Furthermore, thecomposition may comprise between about 0.1-5% or about 0.5-1.5% byweight magnesium stearate as a lubricant.

In one embodiment, the pharmaceutical composition of the invention isabout 50-80% by weight of active ingredient; about 20-40% by weightmicrocrystalline cellulose; about 1-10% by weight croscarmellose sodium;and about 0.1-5% by weight magnesium stearate. In another embodiment,the pharmaceutical composition of the invention is about 60-70% byweight of active ingredient; about 25-35% by weight microcrystallinecellulose; about 2-5% by weight croscarmellose sodium; and about0.5-1.5% by weight magnesium stearate. In one embodiment, thepharmaceutical composition described comprises about 50-200 mg or 50-600mg of SAHA Form I.

A current embodiment of the invention is a solid formulation of SAHAwith microcrystalline cellulose, NF (Avicel Ph 101), sodiumcroscarmellose, NF (AC-Di-Sol) and magnesium stearate, NF, contained ina gelatin capsule. A further embodiment is a pharmaceutical compositioncomprising about 100 mg active ingredient, about 44.3 mg ofmicrocrystalline cellulose, about 4.5 mg of croscarmellose sodium, about1.2 mg of magnesium stearate.

In one embodiment, the pharmaceutical compositions are administeredorally, and are thus formulated in a form suitable for oraladministration, i.e., as a solid or liquid form. Suitable solid oralformulations include for example, tablets, capsules, pills, granules,pellets and the like. Suitable liquid oral formulations include forexample, emulsions, oils and the like. In one embodiment of the presentinvention, the composition is formulated in a capsule. In accordancewith this embodiment, the compositions of the present invention comprisea hard gelatin capsule in addition to the active ingredient and theinert carrier or diluent.

Solid carriers/diluents include, but are not limited to, a gum, a starch(e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose,mannitol, sucrose, dextrose), a cellulosic material (e.g.,microcrystalline cellulose), an acrylate (e.g., polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.

For liquid formulations, pharmaceutically acceptable carriers may benon-aqueous solutions, suspensions, emulsions or oils. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol, andinjectable organic esters such as ethyl oleate. Examples of oils arethose of petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, andfish-liver oil. Suspensions can also include the following components:fixed oils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid (EDTA).

In addition, the compositions may further comprise binders (e.g.,acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g., cornstarch, potato starch, alginic acid,silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodiumstarch glycolate, Primogel), detergents (e.g., Tween 20, Tween 80,Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g.,sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g.,glycerol, polyethylene glycerol), a glidant (e.g., colloidal silicondioxide), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite,butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose,hyroxypropylmethyl cellulose), viscosity increasing agents (e.g.,carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents(e.g., peppermint, methyl salicylate, or orange flavoring),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants(e.g., stearic acid, magnesium stearate, polyethylene glycol, sodiumlauryl sulfate), flow-aids (e.g., colloidal silicon dioxide),plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers(e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate),polymer coatings (e.g., poloxamers or poloxamines), coating and filmforming agents (e.g., ethyl cellulose, acrylates, polymethacrylates)and/or adjuvants.

In one embodiment, the active ingredient is prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

The preparation of pharmaceutical compositions that contain an activecomponent is well understood in the art, for example, by mixing,granulating, or tablet-forming processes. The active therapeuticingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the active agents are mixed with additives customary forthis purpose, such as vehicles, stabilizers, or inert diluents, andconverted by customary methods into suitable forms for administration,such as tablets, coated tablets, hard or soft gelatin capsules, aqueous,alcoholic or oily solutions and the like as detailed above.

In one embodiment, the oral compositions are formulated in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active ingredient calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active ingredient and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals. Incertain embodiments, the dosage unit contains about 600 mg, 550 mg, 500mg, 450 mg, 400 mg, 350 mg, 300 mg, 250 mg, 200 mg, 150 mg, 110 mg, 105mg, 100 mg, 95 mg, 90 mg, 85 mg, 80 mg, 75 mg, 70 mg, 65 mg, 60 mg, 55mg, 50 mg, 45 mg, or 40 mg of active ingredient. In one embodiment, theamount of the active ingredient is about 100 mg.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. In oneembodiment, the pharmaceutical composition is a single capsule, whereinthe amount of the active ingredient is about 100 mg. In one embodiment,the pharmaceutical composition is two capsules, wherein each capsulecontains active ingredient of about 50 mg.

Process of Producing Compositions with Specified Dissolution Rates

The present invention provides a process of producing a crystallinecomposition comprising suberoylanilide hydroxamic acid or apharmaceutically acceptable salt or hydrate thereof as an activeingredient, wherein about 100 mg of the active ingredient has an invitro dissolution profile with a similarity factor (f2) of at least 50to 100 compared to the reference dissolution profile shown in FIG. 2,comprising the steps of:

(a) crystallizing at least two batches of said active ingredient; and

(b) blending at least two batches of the crystalline active ingredientto produce said crystalline composition.

In an alternative process, the crystalline composition is produced bythe following steps:

(a) milling or wet-milling crystalline active ingredient to produce atleast one first batch of crystalline active ingredient;

(b) crystallizing the active ingredient to produce at least one secondbatch of crystalline active ingredient that is larger in size than themilled or wet-milled crystalline active ingredient;

(c) blending at least the first batch with at least the second batch ofcrystalline active ingredient to produce said crystalline composition.

The crystalline composition can then be further processed to produce apharmaceutical composition, wherein the entire active ingredient has anin vitro dissolution profile with a similarity factor (f2) of at least50 to 100 compared to the reference dissolution profile shown in FIG. 1.This can be accomplished, by applying pressure to the crystallinecomposition, for example by encapsulation of the crystallinecompositions with or without excipients. Due to the pressure incurredduring the capsule packing process, breakage occurs on the particles ofthe active ingredient, which affects the particle size distribution,thereby affecting the dissolution rate. The amount of particle breakagecan be affected by capsule density, which is impacted by the tamping pintype and capsule fill weight.

Therefore, in yet another embodiment, the present invention provides aprocess of producing a pharmaceutical composition comprisingsuberoylanilide hydroxamic acid or a pharmaceutically acceptable salt orhydrate thereof as an active ingredient, wherein the entire activeingredient of the pharmaceutical composition has an in vitro dissolutionprofile with a similarity factor (f2) of at least 50 to 100 compared tothe reference dissolution profile shown in FIG. 1, comprising the stepsof:

(a) crystallizing at least two batches of said active ingredient;

(b) blending at least two batches of the crystalline active ingredient;and

(c) producing said pharmaceutical composition from the blended batches.

In one embodiment, the crystalline active ingredient is prepared fromcrystallization of active ingredient or crude active ingredient from anorganic solvent or a mixture of an organic solvent and water. In oneembodiment, the organic solvent is one or more of methanol, ethanol,acetonitrile, isopropanol and acetic acid. In one embodiment, theorganic solvent is ethanol. In one embodiment, the mixture comprisesabout 40-99% ethanol. In one embodiment, the mixture comprises about40-99% ethanol and 60-1% water. In one embodiment, step (c) is performedby encapsulating a portion of the blended crystalline active ingredient.

In an alternative process the pharmaceutical composition is produced bythe following steps:

(a) milling or wet-milling crystalline active ingredient to produce atleast a first batch of crystalline active ingredient;

(b) crystallizing the active ingredient to produce at least a secondbatch of crystalline active ingredient that is larger in size than themilled or wet-milled crystalline active ingredient;

(c) blending at least the first batch with at least the second batch ofcrystalline active ingredient; and

(d) producing said pharmaceutical composition from said blended firstand second batch.

In one embodiment, the first batch of crystalline active ingredient hasa mean particle size of less than about 50 μm and the second batch ofcrystalline active ingredient has a mean particle size more than about130 μm. In another embodiment, the first batch of crystalline activeingredient has a mean particle size of less than about 50 μm and thesecond batch of crystalline active ingredient has a mean particle sizein the range of about 120 to 160 μm. In a particular embodiment, 95% ofthe first batch of crystalline active ingredient is less than about 100μm. In one embodiment, 95% of the second batch crystals are less thanabout 300 μm. In one embodiment, step (d) is performed by encapsulatinga portion of the blended crystalline active ingredient.

In one embodiment, the first batch of crystalline ingredient has a meanparticle size of less than about 60 μm and the second batch ofcrystalline active ingredient has a mean particle size of about 100-250μm. In another embodiment, the first batch of crystalline ingredient hasa mean particle size in the range of about 25 to 45 μm and the secondbatch of crystalline active ingredient has a mean particle size in therange of about 130 to 180 μm.

In one embodiment, the crystalline active ingredient is prepared fromcrystallization of active ingredient or crude active ingredient from anorganic solvent or a mixture of an organic solvent and water. In oneembodiment, the organic solvent is one or more of methanol, ethanol,acetonitrile, isopropanol and acetic acid. In one embodiment, theorganic solvent is ethanol. In one embodiment, the mixture comprisesabout 40-99% ethanol. In one embodiment, the mixture comprises about40-99% ethanol and 60-1% water.

In another embodiment, in step (c), about 40-95% of the second batchcrystalline active ingredient is blended with about 60-5% of the firstbatch milled crystalline active ingredient.

In one embodiment of the above processes the crystallization stepinvolves seeding. In another embodiment of the above processes, theblending ratio is determined by a computer simulation program that usesan encapsulation breakage model and a dissolution model. In oneembodiment, the blending ratio is optimized to obtain a composition witha SAHA dissolution rate profile similar to a reference with adissolution rate profile in FIG. 1.

The invention also provides a process of producing recrystallized activeingredient of suberoylanilide hydroxamic acid or a pharmaceuticallyacceptable salt or hydrate thereof, comprising the steps of:

(a) providing crystalline active ingredient to an organic solvent, wateror a mixture thereof to form a slurry;

(b) heating the slurry to establish 2-30% undissolved crystalline activeingredient; and

(c) cooling the slurry to obtain the recrystallized active ingredient.

In one embodiment, the crystalline active ingredient in step (a) has amean particle size less than about 60 μm.

In another embodiment, the crystalline active ingredient is prepared bythe steps of:

-   -   (i) adding crystalline active ingredient to an organic solvent,        water or mixture thereof to form a seed slurry; and    -   (ii) wet-milling the slurry to achieve wet-milled crystalline        active ingredient.

In another embodiment, the crystalline active ingredient is prepared bythe step of dry-milling crystalline active ingredient. In a furtherembodiment, the crystalline active ingredient is obtained in thepresence of hydroxylamine.

In one embodiment, in step (a), a mixture of 40-99% ethanol and 60-1%water is used. In another embodiment, in step (b), the slurry is heatedto 60-75° C. for about 1-3 hours. In a further embodiment, step (c) isperformed by cooling from between 60 to 75° C. to between 25 to −5° C.in about 15 to 72 hours.

In another embodiment, the processes above further comprises blendingabout 40-95% of recrystallized active ingredient with about 60-5%crystalline active ingredient having mean particle size less than about60 μm.

The invention also provides a process of producing crystalline activeingredient of suberoylanilide hydroxamic acid, comprising the steps of:

(a) providing crystalline active ingredient to a mixture of 40-99%ethanol and 60-1% water to form a slurry;

(b) heating the slurry to establish 2-30% undissolved crystalline activeingredient;

(c) cooling the slurry to obtain the recrystallized active ingredient;and

(d) blending about 40-95% of recrystallized active ingredient with about60-5% crystalline active ingredient having mean particle size less thanabout 60 μm.

The present invention also provides a process of producingrecrystallized active ingredient of suberoylanilide hydroxamic acid or apharmaceutically acceptable salt or hydrate thereof, comprising thesteps of:

(a) adding crystalline active ingredient to an organic solvent or amixture of organic solvent and water to form a slurry;

(b) wet-milling the slurry to achieve crystalline active ingredient withmean particle size less than about 50 μm;

(c) heating the wet-milled slurry to establish about a 5-30% seed bed;and

(d) cooling the slurry to below 25° C. to obtain the recrystallizedactive ingredient.

In one embodiment, in step (a), the mixture contains ethanol and water,in particular, about 40-95% ethanol. In a particular embodiment, amixture of about 1:1 ethanol and water is used. In another particularembodiment, a mixture of about 9:1 ethanol and water is used. In oneembodiment, after the wet-milling step, at least 80-95%, or in anotherembodiment, 95% of the crystalline active ingredient has a particle sizeless than about 100 μm.

In one embodiment, step c) establishes about a 10-20% seed bed. In aparticular embodiment, step c) establishes about a 15% seed bed. In oneembodiment, step c) is achieved by heating the wet-milled slurry at60-70° C. for 1-3 hours. In another embodiment, step c) is achieved byheating the wet-milled slurry at 63-66° C. for about 1-3 hours. In aparticular embodiment, step c) is achieved by heating the wet-milledslurry at 64-65° C. for about 1-3 hours.

In one embodiment, step (d) is performed by cooling from 60-70° C. to25-5° C. in about 15 to 30 hours. In another embodiment, step (d) isperformed by cooling from 64-65° C. to 20-5° C. in about 15 to 30 hours.The cooling process may involve combinations of decreasing thetemperature within a specified period of time, and maintaining thetemperature for a specified period of time.

The above processes may further comprise the step of blendingrecrystallized active ingredient with wet-milled crystalline activeingredient that is produced by steps identical to steps (a) and (b). Thewet-milled crystalline active ingredient can be taken from a portion ofthe wet-milled material of step b). Alternatively, the wet-milledcrystalline active ingredient can be separately prepared according tosteps a) and b). Therefore, the wet-milled crystalline active ingredientmay be produced in the same or different solvent or mixture as comparedto the crystallization conditions of the recrystallized activeingredient. The blending ratio may be determined by computer simulationsoftware. In one embodiment, the blending ratio is 60-80% ofrecrystallized active ingredient and 40-20% wet-milled crystallineactive ingredient. In a particular embodiment, the blending ratio isabout 70% of recrystallized active ingredient and about 30% wet-milledcrystalline active ingredient. In another particular embodiment, in step(a), a mixture of 9:1 or 1:1 ethanol water is used, and the blendingratio is 70% of recrystallized active ingredient and 30% wet-milledcrystalline active ingredient.

The invention also provides a process of producing recrystallized activeingredient of suberoylanilide hydroxamic acid or a pharmaceuticallyacceptable salt or hydrate thereof, comprising the steps of:

(a) providing crystalline active ingredient to an organic solvent, wateror a mixture thereof to a first vessel to form a slurry;

(b) heating the slurry in the first vessel to dissolve substantially allof the crystalline active ingredient;

(c) cooling the contents in step (b) in the first vessel to atemperature that supersaturates the solution.

(d) adding seeds of the crystalline active ingredient to the contents ofstep (c);

(e) aging the contents of step (d) at the same temperature as step (c);

(f) cooling the contents in step (e) to obtain the recrystallized activeingredient.

In one embodiment, step (d) comprises the steps of:

-   -   (i) providing crystalline active ingredient in an organic        solvent, water or mixture thereof to form a seed slurry;    -   (ii) heating and aging the seed slurry to dissolve a portion of        the seeds;    -   (iii) cooling the contents in step (ii) to the same temperature        as in step (c);    -   (iv) transferring the seed slurry in step (iii) to the first        vessel.

In one embodiment, the crystalline active ingredient of step (i) has amean particle size less than about 60 μm. In another embodiment, step(i) is prepared by the steps of:

-   -   (v) adding crystalline active ingredient to an organic solvent,        water or mixture thereof to form a seed slurry;    -   (vi) wet-milling the slurry to achieve wet-milled crystalline        active ingredient.

In another embodiment, step (i) is prepared by the steps of:

-   -   (v) dry-milling crystalline active ingredient;    -   (vi) adding the dry-milled crystalline active ingredient to an        organic solvent, water or mixture thereof to form a seed slurry.

In a further embodiment, after step (vi), further comprises the step ofisolating, washing and drying the wet-milled crystalline activeingredient prior to step (d).

In one embodiment, the crystalline active ingredient of step (a) isobtained in the presence of hydroxylamine. In another embodiment, amixture of 40-99% ethanol and 60-1% water is used in step (a) and (i).In a further embodiment, a mixture of ethanol to water ratio of 49:51 to51:49 is used in step (a) and (i)

In one embodiment, in step (b), the slurry is heated to 60-75° C. underminimum of 15 psig pressure. In another embodiment, in step (b), theslurry is heated to 67-70° C. under minimum of 15 psig pressure.

In one embodiment, in step (c), the contents are cooled to 60-65° C. Inanother embodiment, in step (c), the contents are cooled to 61-63° C.

In one embodiment, in step (ii), the seed slurry is heated to 62-66° C.In another embodiment, in step (ii), the seed slurry is heated to 64-65°C.

In one embodiment, step (f) is performed by cooling from between 60 to70° C., to between 25 to −5° C. in about 15 to 72 hours. In anotherembodiment, step (f) is performed by cooling from between 60 to 64° C.,to between 0 to 10° C. in about 15 to 72 hours.

The present invention also provides a process of producingrecrystallized active ingredient of suberoylanilide hydroxamic acid or apharmaceutically acceptable salt or hydrate thereof, comprising thesteps of:

(a) adding crystalline active ingredient to an organic solvent ormixture of organic solvent and water to form a slurry;

(b) wet-milling the slurry to achieve crystalline active ingredient withmean particle size less than about 50 μm;

(c) heating the wet-milled slurry to 60-70° C. to produce a seed slurry;

(d) providing crystalline active ingredient in an organic solvent ormixture of organic solvent and water;

(e) heating the material in step (d) to dissolve the crystalline activeingredient;

(f) cooling the material in step (e) to obtain a supersaturated solutionwith no nucleation;

(g) transferring the seed slurry in step (c) to the supersaturatedsolution; and

(h) cooling the material in step (g) to below 25° C.

In one embodiment, in step (a) and (d), a mixture is used which containsethanol and water, in particular, about 40-95% ethanol. In a particularembodiment, a mixture of about 1:1 ethanol and water is used. In anotherparticular embodiment, a mixture of about 9:1 ethanol and water is used.The percentage of organic solvent used in step (a) or (d) may be thesame or different. For example, in step (a), about 40-100% ethanol maybe used, while in step d) a mixture of about 1:1 or 9:1 ethanol may beused. In one embodiment, after the wet-milling step, at least 80-95%, or95% of the crystalline active ingredient has a particle size less thanabout 100 μm.

In one embodiment, step c) establishes about a 10-20% seed bed. In aparticular embodiment, step c) establishes about a 15% seed bed. Inanother embodiment, the wet-milled slurry is heated to 63-67° C. Inanother embodiment, the wet-milled slurry is heated to 62-66° C. at20-25 psig, and cooled to 61-63° C. In another embodiment, thewet-milled slurry is heated to dissolve 50% of the seed solid.

In one embodiment, in step (e), heating is at 65-75° C. In a particularembodiment, in step (e), heating is at 67-70° C. In one embodiment, instep (e), the heating is performed under 20-25 psig pressure. In anotherembodiment, in step (f), cooling is at 60-65° C. In yet anotherembodiment, in step (f), cooling is at 61 to 63° C.

In another embodiment, after step (g) and before step (h), the mixtureis aged for 2 hours at 61 to 63° C. In one embodiment, in step (h), thecooling is achieved through three linear steps in 26 hours.

The invention also provides a process of producing recrystallized activeingredient of suberoylanilide hydroxamic acid, comprising the steps of:

-   -   (a) providing crystalline active ingredient to a mixture of        40-99% ethanol and 60-1% water to a first vessel to form a        slurry;    -   (b) heating the slurry in the first vessel to dissolve        substantially all of the crystalline active ingredient;    -   (c) cooling the contents in step (b) in the first vessel to        supersaturate the solution.    -   (d) adding crystalline active ingredient to the contents of step        (c);    -   (e) aging the contents of step (d) at the same temperature as        step (c);    -   (f) cooling the contents in step (e) to obtain the        recrystallized active ingredient.

In a particular embodiment of the above processes, the active ingredientis suberoylanilide hydroxamic acid. In one embodiment, the crystallineactive ingredient is SAHA Form I.

Crystallization with Organic Solvents

In one particular embodiment, the crystalline active ingredient orrecrystallized active ingredient is crystallized from an organic solventor a mixture of water and an organic solvent. The organic solvent may bean alcohol such as methanol, ethanol or isopropanol. In one embodiment,the organic solvent is one or more of methanol, ethanol, acetonitrile,isopropanol and acetic acid. In one embodiment, the organic solvent isethanol.

In another embodiment, the mixture of organic solvent and watercomprises about 1-99% organic solvent and about 99-1% of water. Inanother embodiment, the mixture comprises 40-99% ethanol and 60%-1% ofwater. In one embodiment, the mixture comprises about 15-85% organicsolvent and about 1-15% water. In a particular embodiment, the mixturecomprises about 85% organic solvent and about 15% water. In anotherparticular embodiment, the mixture comprises 1:1 ethanol and water. Inyet another particular embodiment, the mixture comprises 9:1 ethanol andwater. The ratios or percentages of organic solvent to water describedhere are by volume.

In one particular embodiment, the mixture of an organic solvent andwater is an alcohol and water (e.g. methanol/water, ethanol/water,isopropanol/water and the like). However, it should be apparent to aperson skilled in the art that the crystallizations of the methodsdescribed herein can be carried out in any suitable solvents or solventmixtures which may be readily selected by one of skill in the art oforganic synthesis. Such suitable organic solvents, as used herein mayinclude, by way of example and without limitation, chlorinated solvents,hydrocarbon solvents, ether solvents, polar protic solvents and polaraprotic solvents. Suitable halogenated solvents include, but are notlimited to carbon tetrachloride, bromodichloromethane,dibromochloromethane, bromoform, chloroform, bromochloromethane,dibromomethane, butyl chloride, dichloromethane, tetrachloroethylene,trichloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane,1,1-dichloroethane, 1,2-dichloroethane, 2-chloropropane,hexafluorobenzene, 1,2,4-trichlorobenzene, o-dichlorobenzene,chlorobenzene, fluorobenzene, fluorotrichloromethane,chlorotrifluoromethane, bromotrifluoromethane, carbon tetrafluoride,dichlorofluoromethane, chlorodifluoromethane, trifluoromethane,1,2-dichlorotetrafluorethane and hexafluoroethane. Suitable hydrocarbonsolvents include, but are not limited to benzene, cyclohexane, pentane,hexane, toluene, cycloheptane, methylcyclohexane, heptane, ethylbenzene,m-, o-, or p-xylene, octane, indane, nonane. Suitable ether solventsinclude, but are not limited to dimethoxymethane, tetrahydrofuran,1,3-dioxane, 1,4-dioxane, furan, diethyl ether, ethylene glycol dimethylether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether,diethylene glycol diethyl ether, triethylene glycol diisopropyl ether,anisole, or t-butyl methyl ether.

Suitable polar protic solvents include, but are not limited to methanol,ethanol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol,ethylene glycol, 1-propanol, 2-propanol, 2-methoxyethanol, 1-butanol,2-butanol, i-butyl alcohol, t-butyl alcohol, 2-ethoxyethanol, diethyleneglycol, 1-, 2-, or 3-pentanol, neo-pentyl alcohol, t-pentyl alcohol,diethylene glycol monomethyl ether, diethylene glycol monoethyl ether,cyclohexanol, benzyl alcohol, phenol, and glycerol. Suitable polaraprotic solvents include, but are not limited to dimethylformamide(DMF), dimethylacetamide (DMAC),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidinone (NMP),formamide, N-methylacetamide, N-methylformamide, acetonitrile (ACN),dimethylsulfoxide, propionitrile, ethyl formate, methyl acetate,hexachloroacetone, acetone, ethyl methyl ketone, ethyl acetate,isopropyl acetate, t-butyl acetate, sulfolane, N,N-dimethylpropionamide,nitromethane, nitrobenzene, hexamethylphosphoramide.

Methods of Administration

In all of the methods described herein, the pharmaceutical compositionmay be administered orally in a gelatin capsule. The composition may beadministered in unit dosages according to the methods described hereinonce-daily, twice-daily or three times-daily.

The daily administration is then repeated continuously for a period ofseveral days to several years. Oral treatment may continue for betweenone week and the life of the patient. In one embodiment, theadministration takes place for five consecutive days after which timethe patient can be evaluated to determine if further administration isrequired. The administration can be continuous or intermittent, i.e.,treatment for a number of consecutive days followed by a rest period.

The pharmaceutical compositions of the present invention may beadministered at orally at a total daily dose of between 25 to 4000mg/m², for example, about 25 to 1000 mg, 50-1000 mg, 100 mg, 200 mg, 300mg, 400 mg, 600 mg, 800 mg, 1000 mg and the like. Typically the compoundis administered as a single dose when administering up to 400 mg to thepatient. For higher total dosages (i.e., greater than 400 mg), the totalis split into multiple dosages, for example, twice daily, three timesdaily or the like, or spread out over equal periods of time during theday. For example, two doses, e.g., 500 mg each, can be administered 12hours apart to achieve a total dosage of 1000 mg in a day.

In one embodiment, SAHA is administered to the patient at a total dailydosage of 200 mg. In another embodiment, SAHA is administered to thepatient at a total daily dosage of 400 mg. In another embodiment, SAHAis administered to the patient at a total daily dosage of 600 mg.

In one embodiment, the amount of the active ingredient administered tothe patient is less than an amount that would cause toxicity in thepatient. In certain embodiments, the amount of the active ingredientthat is administered to the patient is less than the amount that causesa concentration of the compound in the patient's plasma to equal orexceed the toxic level of the compound. In one embodiment, theconcentration of the active ingredient in the patient's plasma ismaintained at between about 10 nM to about 5000 nM. The optimal amountof the active ingredient that should be administered to the patient inthe practice of the present invention will depend on the particularcompound used and the type of cancer being treated.

Combination Therapy

The methods of the present invention may also comprise initiallyadministering to the subject an antitumor agent so as to render theneoplastic cells in the subject resistant to an antitumor agent andsubsequently administering an effective amount of any of thecompositions of the present invention, effective to selectively induceterminal differentiation, cell growth arrest and/or apoptosis of suchcells.

The antitumor agent may be one of numerous chemotherapy agents such asan alkylating agent, an antimetabolite, a hormonal agent, an antibiotic,colchicine, a vinca alkaloid, L-asparaginase, procarbazine, hydroxyurea,mitotane, nitrosoureas or an imidazole carboxamide. Suitable agents arethose agents that promote depolarization of tubulin. In one embodiment,the antitumor agent is colchicine or a vinca alkaloid; vinblastine orvincristine. In embodiments where the antitumor agent is vincristine,the cells preferably are treated so that they are resistant tovincristine at a concentration of about 5 mg/ml. The treating of thecells to render them resistant to an antitumor agent may be effected bycontacting the cells with the agent for a period of at least 3 to 5days. The contacting of the resulting cells with any of the compoundsabove is performed as described previously. In addition to the abovechemotherapy agents, the compounds may also be administered togetherwith radiation therapy.

Alkylating Agents

Alkylating agents react with nucleophilic residues, such as the chemicalentities on the nucleotide precursors for DNA production. They affectthe process of cell division by alkylating these nucleotides andpreventing their assembly into DNA.

Examples of alkylating agents include, but are not limited to,bischloroethylamines (nitrogen mustards, e.g., chlorambucil,cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracilmustard), aziridines (e.g., thiotepa), alkyl alkone sulfonates (e.g.,busulfan), nitrosoureas (e.g., carmustine, lomustine, streptozocin),nonclassic alkylating agents (altretamine, dacarbazine, andprocarbazine), platinum compounds (carboplastin and cisplatin). Thesecompounds react with phosphate, amino, hydroxyl, sulfihydryl, carboxyl,and imidazole groups.

Under physiological conditions, these drugs ionize and producepositively charged ion that attach to susceptible nucleic acids andproteins, leading to cell cycle arrest and/or cell death. The alkylatingagents are cell cycle phasenonspecific agents because they exert theiractivity independently of the specific phase of the cell cycle. Thenitrogen mustards and alkyl alkone sulfonates are most effective againstcells in the G1 or M phase. Nitrosoureas, nitrogen mustards, andaziridines impair progression from the G1 and S phases to the M phases.Chabner and Collins eds. (1990) “Cancer Chemotherapy: Principles andPractice”, Philadelphia: J B Lippincott.

The alkylating agents are active against wide variety of neoplasticdiseases, with significant activity in the treatment of leukemias andlymphomas as well as solid tumors. Clinically this group of drugs isroutinely, used in the treatment of acute and chronic leukemias;Hodgkin's disease; non-Hodgkin's lymphoma; multiple myeloma; primarybrain tumors; carcinomas of the breast, ovaries, testes, lungs, bladder,cervix, head and neck, and malignant melanoma.

The major toxicity common to all of the alkylating agents ismyelosuppression. Additionally, gastrointestinal adverse effects ofvariable severity occur commonly and various organ toxicities areassociated with specific compounds. Black and Livingston (1990) Drugs39: 489-501; and 39: 652-673.

Antibiotics

Antibiotics (e.g., cytotoxic antibiotics) act by directly inhibiting DNAor RNA synthesis and are effective throughout the cell cycle. Examplesof antibiotic agents include anthracyclines (e.g., doxorubicin,daunorubicin, epirubicin, idarubicin and anthracenedione), mitomycin C,bleomycin, dactinomycin, plicatomycin. These antibiotic agents interferewith cell growth by targeting different cellular components. Forexample, anthracyclines are generally believed to interfere with theaction of DNA topoisomerase II in the regions of transcriptionallyactive DNA, which leads to DNA strand scissions.

Bleomycin is generally believed to chelate iron and forms an activatedcomplex, which then binds to bases of DNA, causing strand scissions andcell death.

The antibiotic agents have been used as therapeutics across a range ofneoplastic diseases, including carcinomas of the breast, lung, stomachand thyroids, lymphomas, myelogenous leukemias, myelomas, and sarcomas.The primary toxicity of the anthracyclines within this group ismyelosuppression, especially granulocytopenia. Mucositis oftenaccompanies the granulocytopenia and the severity correlates with thedegree of myelosuppression. There is also significant cardiac toxicityassociated with high dosage administration of the anthracyclines.

Antimetabolic Agents

Antimetabolic agents (i.e., antimetabolites) are a group of drugs thatinterfere with metabolic processes vital to the physiology andproliferation of cancer cells. Actively proliferating cancer cellsrequire continuous synthesis of large quantities of nucleic acids,proteins, lipids, and other vital cellular constituents.

Many of the antimetabolites inhibit the synthesis of purine orpyrimidine nucleosides or inhibit the enzymes of DNA replication. Someantimetabolites also interfere with the synthesis of ribonucleosides andRNA and/or amino acid metabolism and protein synthesis as well. Byinterfering with the synthesis of vital cellular constituents,antimetabolites can delay or arrest the growth of cancer cells. Examplesof antimetabolic agents include, but are not limited to, fluorouracil(5-FU), floxuridine (5-FUdR), methotrexate, leucovorin, hydroxyurea,thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin,fludarabine phosphate, cladribine (2-CDA), asparaginase, andgemcitabine.

Antimetabolic agents have widely used to treat several common forms ofcancer including carcinomas of colon, rectum, breast, liver, stomach andpancreas, malignant melanoma, acute and chronic leukemia and hair cellleukemia. Many of the adverse effects of antimetabolite treatment resultfrom suppression of cellular proliferation in mitotically activetissues, such as the bone marrow or gastrointestinal mucosa. Patientstreated with these agents commonly experience bone marrow suppression,stomatitis, diarrhea, and hair loss. Chen and Grem (1992) Curr. Opin.Oncol. 4: 1089-1098.

Hormonal Agents

The hormonal agents are a group of drug that regulate the growth anddevelopment of their target organs. Most of the hormonal agents are sexsteroids and their derivatives and analogs thereof, such as estrogens,progestogens, anti-estrogens, androgens, anti-androgens and progestins.These hormonal agents may serve as antagonists of receptors for the sexsteroids to down regulate receptor expression and transcription of vitalgenes. Examples of such hormonal agents are synthetic estrogens (e.g.,diethylstibestrol), antiestrogens (e.g., tamoxifen, toremifene,fluoxymesterol and raloxifene), antiandrogens (bicalutamide, nilutamide,flutamide), aromatase inhibitors (e.g., aminoglutethimide, anastrozoleand tetrazole), luteinizing hormone release hormone (LHRH) analogues,ketoconazole, goserelin acetate, leuprolide, megestrol acetate andmifepristone.

Hormonal agents are used to treat breast cancer, prostate cancer,melanoma and meningioma. Because the major action of hormones ismediated through steroid receptors, 60% receptor-positive breast cancerresponded to first-line hormonal therapy; and less than 10% ofreceptor-negative tumors responded. The main side effect associated withhormonal agents is flare. The frequent manifestations are an abruptincrease of bony pain, erythema around skin lesions, and inducedhypercalcemia.

Specifically, progestogens are used to treat endometrial cancers, sincethese cancers occur in women that are exposed to high levels ofoestrogen unopposed by progestogen.

Antiandrogens are used primarily for the treatment of prostate cancer,which is hormone dependent. They are used to decrease levels oftestosterone, and thereby inhibit growth of the tumor.

Hormonal treatment of breast cancer involves reducing the level ofoestrogen-dependent activation of oestrogen receptors in neoplasticbreast cells. Anti-oestrogens act by binding to oestrogen receptors andprevent the recruitment of coactivators, thus inhibiting the oestrogensignal.

LHRH analogues are used in the treatment of prostate cancer to decreaselevels of testosterone and so decrease the growth of the tumor.

Aromatase inhibitors act by inhibiting the enzyme required for hormonesynthesis. In post-menopausal women, the main source of oestrogen isthrough the conversion of androstenedione by aromatase.

Plant-Derived Agents

Plant-derived agents are a group of drugs that are derived from plantsor modified based on the molecular structure of the agents. They inhibitcell replication by preventing the assembly of the cell's componentsthat are essential to cell division.

Examples of plant derived agents include vinca alkaloids (e.g.,vincristine, vinblastine, vindesine, vinzolidine and vinorelbine),podophyllotoxins (e.g., etoposide (VP-16) and teniposide (VM-26)),taxanes (e.g., paclitaxel and docetaxel). These plant-derived agentsgenerally act as antimitotic agents that bind to tubulin and inhibitmitosis. Podophyllotoxins such as etoposide are believed to interferewith DNA synthesis by interacting with topoisomerase II, leading to DNAstrand scission.

Plant-derived agents are used to treat many forms of cancer. Forexample, vincristine is used in the treatment of the leukemias,Hodgkin's and non-Hodgkin's lymphoma, and the childhood tumorsneuroblastoma, rhabdomyosarcoma, and Wilms' tumor. Vinblastine is usedagainst the lymphomas, testicular cancer, renal cell carcinoma, mycosisfungoides, and Kaposi's sarcoma. Doxetaxel has shown promising activityagainst advanced breast cancer, non-small cell lung cancer (NSCLC), andovarian cancer.

Etoposide is active against a wide range of neoplasms, of which smallcell lung cancer, testicular cancer, and NSCLC are most responsive.

The plant-derived agents cause significant side effects on patientsbeing treated. The vinca alkaloids display different spectrum ofclinical toxicity. Side effects of vinca alkaloids includeneurotoxicity, altered platelet function, myelosuppression, andleukopenia. Paclitaxel causes dose-limiting neutropenia with relativesparing of the other hematopoietic cell lines. The major toxicity of theepipophyllotoxins is hematologic (neutropenia and thrombocytopenia).

Other side effects include transient hepatic enzyme abnormalities,alopecia, allergic reactions, and peripheral neuropathy.

Biologic Agents

Biologic agents are a group of biomolecules that elicit cancer/tumorregression when used alone or in combination with chemotherapy and/orradiotherapy. Examples of biologic agents include immuno-modulatingproteins such as cytokines, monoclonal antibodies against tumorantigens, tumor suppressor genes, and cancer vaccines.

Cytokines possess profound immunomodulatory activity. Some cytokinessuch as interleukin-2 (IL-2, aldesleukin) and interferon-α (IFN-α)demonstrated antitumor activity and have been approved for the treatmentof patients with metastatic renal cell carcinoma and metastaticmalignant melanoma. IL-2 is a T-cell growth factor that is central toT-cell-mediated immune responses. The selective antitumor effects ofIL-2 on some patients are believed to be the result of a cell-mediatedimmune response that discriminate between self and nonself.

Interferon-α includes more than 23 related subtypes with overlappingactivities. IFN-α has demonstrated activity against many solid andhematologic malignancies, the later appearing to be particularlysensitive.

Examples of interferons include, interferon-α, interferon-β (fibroblastinterferon) and interferon-γ (fibroblast interferon). Examples of othercytokines include erythropoietin (epoietin-α), granulocyte-CSF(filgrastin), and granulocyte, macrophage-CSF (sargramostim). Otherimmuno-modulating agents other than cytokines include bacillusCalmette-Guerin, levamisole, and octreotide, a long-acting octapeptidethat mimics the effects of the naturally occuring hormone somatostatin.

Furthermore, the anti-cancer treatment can comprise treatment byimmunotherapy with antibodies and reagents used in tumor vaccinationapproaches. The primary drugs in this therapy class are antibodies,alone or carrying e.g. toxins or chemostherapeutics/cytotoxics to cancercells. Monoclonal antibodies against tumor antigens are antibodieselicited against antigens expressed by tumors, preferably tumor-specificantigens. For example, monoclonal antibody HERCEPTIN® (trastuzumab) israised against human epidermal growth factor receptor2 (HER2) that isoverexpressed in some breast tumors including metastatic breast cancer.Overexpression of HER2 protein is associated with more aggressivedisease and poorer prognosis in the clinic. HERCEPTIN® is used as asingle agent for the treatment of patients with metastatic breast cancerwhose tumors over express the HER2 protein.

Another example of monoclonal antibodies against tumor antigens isRITUXAN® (rituximab) that is raised against CD20 on lymphoma cells andselectively deplete normal and malignant CD20+ pre-B and mature B cells.

RITUXAN is used as single agent for the treatment of patients withrelapsed or refractory low-grade or follicular, CD20+, B cellnon-Hodgkin's lymphoma. MYELOTARG® (gemtuzumab ozogamicin) and CAMPATH®(alemtuzumab) are further examples of monoclonal antibodies againsttumor antigens that may be used.

Tumor suppressor genes are genes that function to inhibit the cellgrowth and division cycles, thus preventing the development ofneoplasia. Mutations in tumor suppressor genes cause the cell to ignoreone or more of the components of the network of inhibitory signals,overcoming the cell cycle checkpoints and resulting in a higher rate ofcontrolled cell growth-cancer. Examples of the tumor suppressor genesinclude Duc-4, NF-1, NF-2, RB, p53, WT1, BRCA1 and BRCA2.

DPC4 is involved in pancreatic cancer and participates in a cytoplasmicpathway that inhibits cell division. NF-1 codes for a protein thatinhibits Ras, a cytoplasmic inhibitory protein. NF-1 is involved inneurofibroma and pheochromocytomas of the nervous system and myeloidleukemia. NF-2 encodes a nuclear protein that is involved in meningioma,schwanoma, and ependymoma of the nervous system. RB codes for the pRBprotein, a nuclear protein that is a major inhibitor of cell cycle. RBis involved in retinoblastoma as well as bone, bladder, small cell lungand breast cancer. P53 codes for p53 protein that regulates celldivision and can induce apoptosis. Mutation and/or inaction of p53 isfound in a wide ranges of cancers. WTI is involved in Wilms' tumor ofthe kidneys. BRCA1 is involved in breast and ovarian cancer, and BRCA2is involved in breast cancer. The tumor suppressor gene can betransferred into the tumor cells where it exerts its tumor suppressingfunctions.

Cancer vaccines are a group of agents that induce the body's specificimmune response to tumors. Most of cancer vaccines under research anddevelopment and clinical trials are tumor-associated antigens (TAAs).TAAs are structures (i.e., proteins, enzymes or carbohydrates) that arepresent on tumor cells and relatively absent or diminished on normalcells. By virtue of being fairly unique to the tumor cell, TAAs providetargets for the immune system to recognize and cause their destruction.Examples of TAAs include gangliosides (GM2), prostate specific antigen(PSA), α-fetoprotein (AFP), carcinoembryonic antigen (CEA) (produced bycolon cancers and other adenocarcinomas, e.g., breast, lung, gastric,and pancreatic cancers), melanoma-associated antigens (MART-1, gap100,MAGE 1,3 tyrosinase), papillomavirus E6 and E7 fragments, whole cells orportions/lysates of autologous tumor cells and allogeneic tumor cells.

Other Therapies

Recent developments have introduced, in addition to the traditionalcytotoxic and hormonal therapies used to treat cancer, additionaltherapies for the treatment of cancer. For example, many forms of genetherapy are undergoing preclinical or clinical trials.

In addition, approaches are currently under development that are basedon the inhibition of tumor vascularization (angiogenesis). The aim ofthis concept is to cut off the tumor from nutrition and oxygen supplyprovided by a newly built tumor vascular system.

In addition, cancer therapy is also being attempted by the induction ofterminal differentiation of the neoplastic cells. Suitabledifferentiation agents include the compounds disclosed in any one ormore of the following references.

a) Polar compounds (Marks et al (1987); Friend, C., Scher, W., Holland,J. W., and Sato, T. (1971) Proc. Natl. Acad. Sci. (USA) 68: 378-382;Tanaka, M., Levy, J., Terada, M., Breslow, R., Rifkind, R. A., andMarks, P. A. (1975) Proc. Natl. Acad. Sci. (USA) 72: 1003-1006; Reuben,R. C., Wife, R. L., Breslow, R., Rifkind, R. A., and Marks, P. A. (1976)Proc. Natl. Acad. Sci. (USA) 73: 862-866);

b) Derivatives of vitamin D and retinoic acid (Abe, E., Miyaura, C.,Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshika, S., andSuda, T. (1981) Proc. Natl. Acad. Sci. (USA) 78: 4990-4994; Schwartz, E.L., Snoddy, J. R., Kreutter, D., Rasmussen, H., and Sartorelli, A. C.(1983) Proc. Am. Assoc. Cancer Res. 24: 18; Tanenaga, K., Hozumi, M.,and Sakagami, Y. (1980) Cancer Res. 40: 914-919);

c) Steroid hormones (Lotem, J. and Sachs, L. (1975) Int. J. Cancer 15:731-740);

d) Growth factors (Sachs, L. (1978) Nature (Lond.) 274: 535, Metcalf, D.(1985) Science, 229: 16-22);

e) Proteases (Scher, W., Scher, B. M., and Waxman, S. (1983) Exp.Hematol. 11: 490-498; Scher, W., Scher, B. M., and Waxman, S. (1982)Biochem. & Biophys. Res. Comm. 109: 348-354);

f) Tumor promoters (Huberman, E. and Callaham, M. F. (1979) Proc. Natl.Acad. Sci. (USA) 76: 1293-1297; Lottem, J. and Sachs, L. (1979) Proc.Natl. Acad. Sci. (USA) 76: 5158-5162); and

g) inhibitors of DNA or RNA synthesis (Schwartz, E. L. and Sartorelli,A. C. (1982) Cancer Res. 42: 2651-2655, Terada, M., Epner, E., Nudel,U., Salmon, J., Fibach, E., Rifkind, R. A., and Marks, P. A. (1978)Proc. Natl. Acad. Sci. (USA) 75: 2795-2799; Morin, M. J. and Sartorelli,A. C. (1984) Cancer Res. 44: 2807-2812; Schwartz, E. L., Brown, B. J.,Nierenberg, M., Marsh, J. C., and Sartorelli, A. C. (1983) Cancer Res.43: 2725-2730; Sugano, H., Furusawa, M., Kawaguchi, T., and Ikawa, Y.(1973) Bibl. Hematol. 39: 943-954; Ebert, P. S., Wars, I., and Buell, D.N. (1976) Cancer Res. 36: 1809-1813; Hayashi, M., Okabe, J., and Hozumi,M. (1979) Gann 70: 235-238),

The combination of the pharmaceutical compositions of this invention andany of the anti-cancer agents described above and their use thereof, arewithin the scope of the present invention.

Methods of Treatment

The present invention also provides a method of treating a patienthaving a tumor characterized by proliferation of neoplastic cells whichcomprises administering to the patient an effective amount of any of thecompositions of the present invention above, effective to selectivelyinduce terminal differentiation of such neoplastic cells and therebyinhibit their proliferation.

The method of the present invention is intended for the treatment ofhuman patients with cancer. However, it is also likely that the methodwould be effective in the treatment of cancer in other mammals. Cancerincludes but is not limited to any cancer caused by the proliferation ofneoplastic cells, such as lung cancer, acute lymphoid myeloma, Hodgkinslymphoma, non-Hodgkins lymphoma, bladder melanoma, renal carcinoma,breast carcinoma, prostate carcinoma, ovarian carcinoma or colorectalcarcinoma.

The invention is illustrated in the examples in the Experimental DetailsSection which follows. This section is set forth to aid in anunderstanding of the invention but is not intended to, and should not beconstrued to limit in any way the invention as set forth in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS SECTION Example 1 Synthesis of SAHA Form I

SAHA Form I can be synthesized according to the method outlined below,or by any modification and variants thereof.

Synthesis of SAHA Step 1—Synthesis of Suberanilic Acid

In a 22 L flask was placed 3,500 g (20.09 moles) of suberic acid, andthe acid melted with heat. The temperature was raised to 175° C., andthen 2,040 g (21.92 moles) of aniline was added. The temperature wasraised to 190° C. and held at that temperature for 20 minutes. The meltwas poured into a Nalgene tank that contained 4,017 g of potassiumhydroxide dissolved in 50 L of water. The mixture was stirred for 20minutes following the addition of the melt. The reaction was repeated atthe same scale, and the second melt was poured into the same solution ofpotassium hydroxide. After the mixture was thoroughly stirred, thestirrer was turned off, and the mixture was allowed to settle. Themixture was then filtered through a pad of Celite (4,200 g) (the productwas filtered to remove the neutral by-product (from attack by aniline onboth ends of suberic acid). The filtrate contained the salt of theproduct, and also the salt of unreacted suberic acid. The mixture wasallowed to settle because the filtration was very slow, taking severaldays.). The filtrate was acidified using 5 L of concentratedhydrochloric acid; the mixture was stirred for one hour, and thenallowed to settle overnight. The product was collected by filtration,and washed on the funnel with deionized water (4×5 L). The wet filtercake was placed in a 72 L flask with 44 L of deionized water, themixture heated to 50° C., and the solid isolated by a hot filtration(the desired product was contaminated with suberic acid which has a muchgreater solubility in hot water. Several hot triturations were done toremove suberic acid. The product was checked by NMR [D₆DMSO] to monitorthe removal of suberic acid). The hot trituration was repeated with 44 Lof water at 50° C. The product was again isolated by filtration, andrinsed with 4 L of hot water. It was dried over the weekend in a vacuumoven at 65° C. using a Nash pump as the vacuum source (the Nash pump isa liquid ring pump (water) and pulls a vacuum of about 29 inch ofmercury. An intermittent argon purge was used to help carry off water);4,182.8 g of suberanilic acid was obtained.

The product still contained a small amount of suberic acid; thereforethe hot trituration was done portionwise at 65° C., using about 300 g ofproduct at a time. Each portion was filtered, and rinsed thoroughly withadditional hot water (a total of about 6 L). This was repeated to purifythe entire batch. This completely removed suberic acid from the product.The solid product was combined in a flask and stirred with 6 L ofmethanol/water (1:2), and then isolated by filtration and air dried onthe filter over the week end. It was placed in trays and dried in avacuum oven at 65° C. for 45 hours using the Nash pump and an argonbleed. The final product has a weight of 3,278.4 g (32.7% yield).

Step 2—Synthesis of Methyl Suberanilate

To a 50 L flask fitted with a mechanical stirrer, and condenser wasplaced 3,229 g of suberanilic acid from the previous step, 20 L ofmethanol, and 398.7 g of Dowex 50WX2-400 resin. The mixture was heatedto reflux and held at reflux for 18 hours. The mixture was filtered toremove the resin beads, and the filtrate was taken to a residue on arotary evaporator.

The residue from the rotary evaporator was transferred into a 50 L flaskfitted with a condenser and mechanical stirrer. To the flask was added 6L of methanol, and the mixture heated to give a solution. Then 2 L ofdeionized water was added, and the heat turned off. The stirred mixturewas allowed to cool, and then the flask was placed in an ice bath, andthe mixture cooled. The solid product was isolated by filtration, andthe filter cake was rinsed with 4 L of cold methanol/water (1:1). Theproduct was dried at 45° C. in a vacuum oven using a Nash pump for atotal of 64 hours to give 2,850.2 g (84% yield) of methyl suberanilate,CSL Lot # 98-794-92-3 1.

To a 50 L flask with a mechanical stirrer, thermocouple, and inlet forinert atmosphere was added 1,451.9 g of hydroxylamine hydrochloride, 19L of anhydrous methanol, and a 3.93 L of a

Step 3—Synthesis of Crude SAHA

30% sodium methoxide solution in methanol. The flask was then chargedwith 2,748.0 g of methyl suberanilate, followed by 1.9 L of a 30% sodiummethoxide solution in methanol. The mixture was allowed to stir for 16hr and 10 minutes. Approximately one half of the reaction mixture wastransferred from the reaction flask (flask 1) to a 50 L flask (flask 2)fitted with a mechanical stirrer. Then 27 L of deionized water was addedto flask 1 and the mixture was stirred for 10 minutes. The pH was takenusing a pH meter; the pH was 11.56. The pH of the mixture was adjustedto 12.02 by the addition of 100 ml of the 30% sodium methoxide solutionin methanol; this gave a clear solution (the reaction mixture at thistime contained a small amount of solid. The pH was adjusted to give aclear solution from which the precipitation the product would beprecipitated). The reaction mixture in flask 2 was diluted in the samemanner; 27 L of deionized water was added, and the pH adjusted by theaddition of 100 ml of a 30% sodium methoxide solution to the mixture, togive a pH of 12.01 (clear solution).

The reaction mixture in each flask was acidified by the addition ofglacial acetic acid to precipitate the product. Flask 1 had a final pHof 8.98, and Flask 2 had a final pH of 8.70. The product from bothflasks was isolated by filtration using a Buchner funnel and filtercloth. The filter cake was washed with 15 L of deionized water, and thefunnel was covered and the product was partially dried on the funnelunder vacuum for 15.5 hr. The product was removed and placed into fiveglass trays. The trays were placed in a vacuum oven and the product wasdried to constant weight. The first drying period was for 22 hours at60° C. using a Nash pump as the vacuum source with an argon bleed. Thetrays were removed from the vacuum oven and weighed. The trays werereturned to the oven and the product dried for an additional 4 hr and 10minutes using an oil pump as the vacuum source and with no argon bleed.The material was packaged in double 4-mill polyethylene bags, and placedin a plastic outer container. The final weight after sampling was 2633.4g (95.6%).

Step 4—Preparation of SAHA Form I by Recrystallization of Crude SAHA

The crude SAHA was recrystallized from methanol/water. A 50 L flask witha mechanical stirrer, thermocouple, condenser, and inlet for inertatmosphere was charged with the crude SAHA to be crystallized (2,525.7g), followed by 2,625 ml of deionized water and 15,755 nu of methanol.The material was heated to reflux to give a solution. Then 5,250 ml ofdeionized water was added to the reaction mixture. The heat was turnedoff, and the mixture was allowed to cool. When the mixture had cooledsufficiently so that the flask could be safely handled (28° C.), theflask was removed from the heating mantle, and placed in a tub for useas a cooling bath. Ice/water was added to the tub to cool the mixture to−5° C. The mixture was held below that temperature for 2 hours. Theproduct was isolated by filtration, and the filter cake washed with 1.5L of cold methanol/water (2:1). The funnel was covered, and the productwas partially dried under vacuum for 1.75 hr. The product was removedfrom the funnel and placed in 6 glass trays. The trays were placed in avacuum oven, and the product was dried for 64.75 hr at 60° C. using aNash pump as the vacuum source, and using an argon bleed. The trays wereremoved for weighing, and then returned to the oven and dried for anadditional 4 hours at 60° C. to give a constant weight. The vacuumsource for the second drying period was a oil pump, and no argon bleedwas used. The material was packaged in double 4-mill polyethylene bags,and placed in a plastic outer container. The final weight after samplingwas 2,540.9 g (92.5%).

In other experiments, crude SAHA was crystallized using the followingconditions:

TABLE 1 SAHA Crystallization Conditions Solvent Water Agitation Time(hr) Methanol — Off 2 Methanol — On 72 Ethanol — On 72 Isopropanol — Off72 Ethanol 15% On 2 Methanol 15% Off 72 Ethanol 15% Off 72 Ethanol 15%On 72 Methanol 15% On 72

All these reaction conditions produced SAHA Polymorph I.

Example 1A Production of SAHA Form I

Step 1 8-anilino-8-oxooctanoic acid; suberanilic acid (Compound 3)

Suberic acid (Compound 1, 174.2 g, 1.0 mole), aniline (Compound 2,85.8-94.9 g), and toluene (0.1-0.2 L) are combined, heated to reflux andrefluxed for a minimum of 60 hours. The reaction is quenched at refluxby adjusting the pH to ≧11 with 10% sodium hydroxide solution. Theaqueous phase is separated. The organic layer is combined with toluene(0.11-0.13 L) and water (0.3-0.4 L), and the aqueous layer is separated.The aqueous layers from the extractions and toluene (0.11-0.13 L) arecombined, settled, and then separated. The aqueous layer is extractedtwice with toluene (0.2-0.3 L) at 60-70° C. The aqueous layer isadjusted at 20-30° C. to a pH of 5.8-6.2, using hydrochloric acid and10% sodium hydroxide solution as needed. The batch is filtered, washedwith chilled water (0.2-0.3 L) and then washed with chilled isopropanol.The wet cake is dried at a maximum of 65° C. under vacuum to yieldsuberanilic acid.

Step 2 methyl 8-anilino-8-oxooctanoate; methyl suberanilate (Compound 4)

Suberanilic acid (Compound 3, 249.3 g, 1.0 mole) and methanol (0.4-0.5L) are combined and heated to 45-55° C. The pH is adjusted to ≦2 usinghydrochloric acid, and the batch temperature is maintained at 45-55° C.until the reaction is complete. The reaction is quenched with deionizedwater (0.1-0.2 L). The batch is cooled to 25-30° C. and seeded to inducecrystallization, and then cooled to 0-10° C. The batch is filtered, andthe cake washed with a 50:50 (v/v) methanol/water solution (0.28-0.34 L)at 0-10° C. The wet cake is dried at a maximum of 46° C. under vacuum toyield methyl suberanilate.

Step 3 N-hydroxy-N′ -phenyloctanediamide; vorinostat (Compound 5)

Methyl suberanilate (Compound 4, 263.3 g, 1.0 mole) and 2M hydroxylaminefreebase solution (0.8-1.0 L) are combined. While maintaining the batchat a maximum of 20° C., the apparent pH is adjusted to ≧10.5 with sodiummethoxide in methanol as needed. While maintaining the batch at maximum20° C. and apparent pH≧10.5 using sodium methoxide in methanol, thebatch is aged. During the age, hydroxylamine freebase solution (0.5-0.6L) is added, and the batch is maintained at maximum 20° C. and apparentpH≧10.5 until the reaction is complete. The reaction is quenched byadding the batch to water (0.9-1.1 L) while maintaining the batchtemperature between 20-35° C., and the water content of the batch isadjusted to 35-45%. The pH is adjusted to 8.8-9.2 using glacial aceticacid and sodium carbonate as needed. The batch is cooled to 0-10° C.over 5-10 hours. The batch is filtered and the cake washed with 55:45(v/v) methanol/water (0.45-0.6 L) at 0-10° C. The wet cake is vacuumconditioned until the water content is ≦35%.

The vorinostat crude (264.32 g, 1.0 mole) wet cake is combined withdenatured ethanol (1308-1599 g) and water (167-204 g). Hydroxylaminehydrochloride (>9 mEquiv) and sodium methoxide in methanol (>9 mEquiv)are added to the slurry, and the batch is heated to 70-80° C. Thesolution is filtered and then crystallized by slowly cooling to 0-10° C.The batch is filtered and the cake washed with cold 4:1 (v/v) denaturedethanol/water. The wet cake is dried at a maximum of 45° C. undervacuum.

Step 4 N-hydroxy-N′-phenyloctanediamide—vorinostat-fine (Compound 6)

Vorinostat (Compound 5, 264.3 g, 1.0 mole) is slurried in a 50:50 (v/v)ethanol/water solution (minimum 2.8 L). The vorinostat slurry iswet-milled to a mean size of 25-45 μm while maintaining the batchtemperature at 7-30° C. The final slurry is filtered and the wet cake iswashed with 0-40° C. water (minimum 0.8 L). The wet cake is dried at amaximum of 55° C. under vacuum to a maximum water content of 0.2% (w/w)to yield vorinostat-fine drug substance.

Step 5 N-hydroxy-N′-phenyloctanediamide—vorinostat-coarse (Compound 7)

Vorinostat (Compound 5, 264.3 g, 1.0 mole) is slurried in a 50:50 (v/v)ethanol/water solution (4.9-5.5 L). Under a minimum of 15 psig pressure,the slurry is heated to 65-70° C. to dissolve and then cooled to 60-64°C. A seed slurry is transferred into the batch while maintaining thebatch temperature. The batch is aged for a minimum of 2 hours at 61-63°C. The batch is cooled in three steps by controlling the jackettemperature: (1) to 55° C. at 0.35-0.78° C./hour, (2) to 45° C. at0.83-2.00° C./hour, and (3) to −5 to 25° C. at 2.00-4.44° C./hour. Thefinal slurry is aged at −5 to 25° C. for about 1 hour and then filtered.The wet cake is washed with water (minimum 0.8 L). The wet cake is driedat a maximum of 55° C. under vacuum to yield vorinostat-fine drugsubstance.

The seed slurry is prepared by combining vorinostat-fine dry cake(97.8-116.3 g, 0.37-0.44 mol) and 50:50 (v/v) ethanol/water solution(1.0-1.2 L). Under a minimum of 15 psig pressure, the seed slurry isheated to 62-66° C., aged for about 0.5 hours and then cooled to 60-64°C.

Example 2 Generation of Wet-Milled Small Particles in 1:1 Ethanol/Water

The SAHA Polymorph I crystals were suspended in 1:1 (by volume)EtOH/water solvent mixture at a slurry concentration ranging from 50mg/gram to 150 mg/gram (crystal/solvent mixture). The slurry was wetmilled with IKA-Works Rotor-Stator high shear homogenizer model T50 withsuperfine blades at 20-30 m/s, until the mean particle size of SAHA wasless than 50 μm and 95% less than 100 μm, while maintaining thetemperature at room temperature. The wet-milled slurry was filtered andwashed with the 1:1 EtOH/water solvent mixture at room temperature. Thewet cake was then dried at 40° C. The final mean particle size of thewet-milled material was less than 50 μm as measured by the Microtracmethod below.

Particle size was analyzed using an SRA-150 laser diffraction particlesize analyzer, manufactured by Microtrac Inc. The analyzer was equippedwith an ASVR (Automatic Small Volume Recirculator). 0.25 wt % lecithinin ISOPAR G was used as the dispersing fluid. Three runs were recordedfor each sample and an average distribution was calculated. Particlesize distribution (PSD) was analyzed as a volume distribution. The meanparticle size and 95%<values based on volume were reported.

Example 2A Large Scale Generation of Wet-Milled Small Particles in 1:1Ethanol/Water

56.4 kg SARA Polymorph I crystals were charged to 610 kg (10.8 kgsolvent per kg SAHA) of a 50% vol/vol solution of 200 proof punctiliousethanol and water (“50/50 EtOH/Water”) at 20-25° C. The slurry (˜700L)was recirculated through an IKA Works wet-mill set with super-finegenerators until reaching a steady-state particle size distribution. Theconditions were: DR3-6, 23 m/s rotor tip speed, 30-35 Lpm, 3 gen, ˜70turnovers (a turnover is one batch volume passed through one gen).

${{{Approx}.\mspace{14mu} {Mill}}\mspace{14mu} {Time}\mspace{14mu} ({hr})} = \frac{70 \times {Batch}\mspace{14mu} {Volume}\mspace{14mu} (L)}{\begin{matrix}{{Natural}\mspace{14mu} {Draft}\mspace{14mu} {of}\mspace{14mu} {Mill}\mspace{14mu} ({Lpm}) \times} \\{\# \mspace{14mu} {of}\mspace{14mu} {Generators} \times 60}\end{matrix}}$

The wet cake was filtered, washed with water (total 3 kg/kg, ˜170 kg)and vacuum dried at 40-45° C. The dry cake was then sieved (595 μmscreen) and packed as the “Fine API”.

Example 3 Growth of Large Crystals of Mean Particle Size 150 μm in 1:1Ethanol/Water

25 grams of SAHA Polymorph I crystals and 388 grams of 1:1 Ethanol/watersolvent mixture were charged into a 500 ml jacketed resin kettle with aglass agitator. The slurry was wet milled to a particle size less than50 μm at room temperature following the steps of Example 2. Thewet-milled slurry was heated to 65° C. to dissolve ˜85% of the solid.The heated slurry was aged at 65° C. for 1-3 hours to establish a ˜15%seed bed. The slurry was mixed in the resin kettle under 20 psigpressure, and at an agitator speed range of 400-700 rpm.

The batch was then cooled slowly to 5° C.: 65 to 55° C. in 10 hours, 55to 45° C. in 10 hours, 45 to 5° C. in 8 hours. The cooled batch was agedat 5° C. for one hour to reach a target supernatant concentration ofless than 5 mg/g, in particular, 3 mg/g. The batch slurry was filteredand washed with 1:1 EtOH/water solvent mixture at 5° C. The wet cake wasdried at 40° C. under vacuum. The dry cake had a final particle size of˜150 μm with 95% particle size<300 μm according to the Microtrac method.

Example 3A Growth of Large Crystals in 1:1 Ethanol/Water

13.4 kg vorinostat and 134 kg of a 1:1 (v/v) solution of ethanol andwater are combined. The resulting slurry is wet-milled to a mean size of95%<100 μm. An additional 20 kg of the 1:1 solution is added and thebatch is heated under 20 psig nitrogen pressure to 69-71° C. and agedfor 3 hours to establish a seed bed. While maintaining 20 psig pressure,the batch is cooled to 64-66° C. over 8 hours; to 59-61° C. over 4hours; to 49-51° C. over 4 hours; then to 14-16° C. over 6 hours. Thebatch is filtered and the cake is washed with a total of approximately80 kg water. The batch is vacuum dried at maximum of 55° C.

Example 4 Growth of Large Crystals with Mean Particle Size of 140 μm in1:1 Ethanol/Water

7.5 grams of SAHA Polymorph I crystals and 70.7 grams of 1:1 EtOH/watersolvent mixture were charged into a seed preparation vessel (500-mljacketed resin kettle). The seed slurry was wet milled to a particlesize less than 50 μm at room temperature following the steps of Example2 above. The seed slurry was heated to 63-67° C. and aged over 30minutes to 2 hours.

In a separate crystallizer (1-liter jacketed resin kettle), 17.5 gramsof SAHA Polymorph I crystals and 317.3 grams of 1:1 EtOH/water solventmixture were charged. The crystallizer was heated to 67-70° C. todissolve all solid SAHA crystals first, and then was cooled to 60-65° C.to keep a slightly supersaturated solution.

The seed slurry from the seed preparation vessel was transferred to thecrystallizer. The slurry was mixed in the resin kettle under 20 psigpressure, and at an agitator speed range similar to that in Example 3.The batch slurry was cooled slowly to 5° C. according to the coolingprofile in Example 3. The batch slurry was filtered and washed with 1:1EtOH/water solvent mixture at 5° C. The wet cake was dried at 40° C.under vacuum. The dry cake had a final particle size of about 140 μmwith 95% particle size<280 μm.

Example 4A Large Scale Growth of Large Crystals in 1:1 Ethanol/Water

21.7 kg of the “Fine API” dry cake from Example 2A (28.6% of total, 0.40Equiv. w.r.t basis) and 213 kg of 50/50 EtOH/Water solution (3.93 kgsolvent/kg SAHA basis) was charged to Vessel #1—the Seed PreparationTank. 54.2 kg of SAHA Polymorph I crystals (71.4% of total, 1.00 Equiv,Basis) and 990 kg 50/50 EtOH/Water (18.24 kg solvent/kg SAHA basis) wascharged to Vessel #2—the Crystallizer. The Crystallizer was pressurizedto 20-25 psig and the contents heated to 67-70° C. while maintaining thepressure to fully dissolve the crystalline SAHA. The contents were thencooled to 61-63° C. to supersaturate the solution. During the agingprocess in the Crystallizer, the Seed Prep Tank was pressurized to 20-25psig, the seed slurry was heated to 64° C., aged for 30 minutes whilemaintaining the pressure to dissolve ˜½ of the seed solids, and thencooled to 61-63° C.

The hot seed slurry was rapidly transferred from the Seed Prep Tank tothe Crystallizer (no flush) while maintaining both vessel temperatures.The nitrogen pressure in the Crystallizer was re-established to 20-25psig and the batch was aged for 2 hours at 61-63° C. The batch wascooled to 5° C. in three linear steps over 26 hours: (1) from 62° C. to55° C. over 10 hours; (2) from 55° C. to 45° C. over 6 hours; and (3)from 45° C. to 5° C. over 10 hours. The batch was aged for 1 hr and thenthe wet cake was filtered and washed with water (total 3 kg/kg SAHA,˜163 kg), and vacuum dried at 40-45° C. The dry cake from thisrecrystallization process is packed-out as the “Coarse API”. Coarse APIand Fine API were blended at a 70/30 ratio.

SAHA Polymorph I crystals in the Crystallizer can be prepared by adding8.7 kg SAHA to 72 kg of a 9:1 (v/v) solution of ethanol and water. 25 gof hydroxylamine hydrochloride is charged followed by 350 g of a 1Naqueous solution of sodium hydroxide. The resulting slurry is heated to69.5-71.5° C. and aged for 45 minutes to dissolve the batch and reducethe levels of the O-suberanilic SAHA impurity. The batch is cooled to 4°C. over 2 hours and aged at 0-10° C. for 2 hrs. The batch is filteredand the cake is washed with a total of approximately 60 kg water. Thebatch is vacuum dried at maximum of 55° C. to produce 8.0 kg ofvorinostat.

Example 5 Generation of Wet-Milled Small Particles Batch 288

SAHA Polymorph I crystals were suspended in ethanolic aqueous solution(100% ethanol to 50% ethanol in water by volume) at a slurryconcentration ranging from 50 mg/gram to 150 mg/gram (crystal/solventmixture). The slurry was wet milled with IKA-Works Rotor-Stator highshear homogenizer model T50 with superfine blades at 20-35 m/s, untilthe mean particle size of SAHA was less than 50 μm and 95% less than 100μm, while maintaining the temperature at room temperature. Thewet-milled slurry was filtered and washed with EtOH/water solventmixture at room temperature. The wet cake was then dried at 40° C. Thefinal mean particle size of the wet-milled material was less than 50 μmas measured by the Microtrac method as described before.

Example 6 Growth of Large Crystals Batch 283

24 grams of SAHA Polymorph I crystals and 205 ml of 9:1 Ethanol/watersolvent mixture were charged into a 500 ml jacketed resin kettle with aglass agitator. The slurry was wet milled to a particle size less than50 μm at room temperature following the steps of Example 1. Thewet-milled slurry was heated to 65° C. to dissolve ˜85% of the solid.The heated slurry was aged at 64-65° C. for 1-3 hours to establish a˜15% seed bed. The slurry was mixed at an agitator speed range of100-300 rpm.

The batch was then cooled to 20° C. with one heat-cool cycle: 65° C. to55° C. in 2 hours, 55° C. for 1 hour, 55° C. to 65° C. over ˜30 minutes,age at 65° C. for 1 hour, 65° C. to 40° C. in 5 hours, 40° C. to 30° C.in 4 hours, 30° C. to 20° C. over 6 hours. The cooled batch was aged at20° C. for one hour. The batch slurry was filtered and washed with 9:1EtOH/water solvent mixture at 20° C. The wet cake was dried at 40° C.under vacuum. The dry cake had a final particle size of ˜150 μm with 95%particle size <300 μm per Microtrac method.

Example 7 X-Ray Powder Diffraction Analysis

X-ray Powder Diffraction analysis was performed on SAHA Form I obtainedin accordance with Examples 1-6, and on SAHA Form II-V prepared bymethods detailed in Table 2 below.

TABLE 2 SAHA Samples analyzed by X-ray Powder Diffraction SAHA SampleReference Method SAHA Form I — Examples 1-6 SAHA Form II U.S. Pat. No.5,369,108 SAHA was dissolved in EtOAc/THF (3/1). The Columns 25-26solutions were passed through a plug of silica gel Procedures A, C, Dusing EtOAc/THF (3/1). Fractions were collected and concentrated. Thesolid appeared pink. SAHA Form III U.S. Pat. No. 5,369,108 SAHA wasdissolved in methanol, filtered via Columns 25-26 celite, andconcentrated on the rotovap to dryness. Procedure B The residues wereslurried with hexanes and filtered. The solids appeared pink. SAHA FormIV Mai et al OPPI Briefs SAHA was recrystallized from acetonitrile.(2001)Vol 33(4), 391-394 SAHA Form V Stowell et al J. Med. To a mixtureof SAHA (4.0 g) in anhydrous Chem. (1995), 38(8), methanol (15 mL) wasadded NaOMe (10.7 mL, 1411-1413 4.37 M, 47 mmol). The solution becamehomogeneous, but solid formed after about 5 minutes. The mixture wasstirred for 15 min, and then 100 ml of water was added followed by slowaddition of glacial acetic acid (3.77 mL, 4.0 g). The crystalline solidwas collected and washed with water (2 × 75 mL). The solid was driedunder high vaccum overnght yielding 3.85 g (96% recovery) of anoff-white solid.

X-Ray Diffraction Analysis:

The samples were analyzed on a Siemens D500 Automated PowderDiffractometer (Instrument ID No. LD-301-4), which is operated accordingto Standard Operating Procedure EQ-27, Rev. 12, in accordance with themanufacturer's instructions. The Diffractometer is equipped with agraphite monochromator and a Cu (λ=1.54 A) X-ray source operated at 50kV, 40 mA. Two-theta calibration is performed using an NBS mica standard(SRM675). The samples were analyzed using the following instrumentparameters:

Measuring Range: 4-40 2 theta

Step Width: 0.05 Å

Measuring Time per Step: 1.2 seconds

Sample preparation was performed according to Standard OperatingProcedure MIC-7, Rev. 2 (Section 3.1.2), in accordance with themanufacturer's instructions, using a zero background sample plate (#1).The samples were processed following a light mortar and pestle grind toensure homogeneity.

FIG. 7A-E depicts the X-ray diffractograms for SAHA Forms I-V. Thecorresponding data for the X-ray diffractograms is presented in Tables3-7 below:

TABLE 3 SAHA Form I Peak 2Theta (deg) D (Å) 1 8.97 9.86159 2 9.37 9.43 317.46 5.07 4 19.41 4.57 5 20.04 4.43 6 23.96 3.71 7 24.44 3.64 8 24.763.59 9 24.96 3.56 10 27.96 3.19 11 43.29 2.08

TABLE 4 SAHA Form II 2Theta Peak (deg) D (Å) 1 5.12 17.24 2 5.46 16.15 37.48 11.8 4 7.72 11.44 5 8.15 18.84 6 8.72 10.13 7 9.21 9.59 8 10.918.09 9 12.38 7.14 10 13.55 6.52 11 17.31 5.12 12 18.22 4.86 13 18.864.70 14 19.32 4.59 15 19.88 4.46 16 20.76 4.27 17 21.20 4.19 18 21.724.09 19 22.07 4.02 20 22.88 3.88 21 23.36 3.80 22 23.79 3.73 23 24.163.68 24 24.66 3.61 25 25.75 3.46 26 26.92 3.31 27 27.56 3.23 28 27.883.20 29 28.53 3.12 30 30.68 2.91 31 40.21 2.24 32 42.80 2.11 33 43.162.09

TABLE 5 SAHA Form III 2Theta Peak (deg) D (Å) 1 10.10 8.75 2 12.13 7.293 13.83 6.40 4 15.11 5.86 5 17.65 5.02 6 18.54 4.78 7 18.80 4.71 8 19.604.52 9 20.18 4.40 10 20.90 4.25 11 21.69 4.10 12 23.81 3.73 13 24.543.62 14 25.04 3.55 15 25.36 3.51 16 26.10 3.41 17 26.80 3.32 18 35.622.51 19 37.12 2.42 20 40.92 2.20 21 42.43 2.13 22 44.83 2.02

TABLE 6 SAHA Form IV Peak 2Theta (deg) D (Å) 1 8.84 9.99 2 9.25 9.55 311.00 8.04 4 12.44 7.11 5 17.38 5.10 6 19.37 4.58 7 19.93 4.45 8 22.363.97 9 22.89 3.88 10 23.83 3.73 11 24.24 3.67 12 24.80 3.59 13 25.803.45 14 26.96 3.30 15 27.84 3.20 16 28.39 3.14

TABLE 7 SAHA Form V Peak 2Theta (deg) D (Å) 1 5.08 17.39 2 9.20 9.60 310.07 8.77 4 12.13 7.29 5 15.09 5.86 6 17.65 5.02 7 19.32 4.59 8 19.804.48 9 20.16 4.41 10 20.87 4.25 11 21.67 4.10 12 24.56 3.62 13 25.253.52 14 26.10 3.41 15 35.62 2.51 16 37.12 2.42 17 40.90 2.20 18 41.782.16 19 42.42 2.13 20 44.82 2.02

The X-ray powder diffraction pattern of SAHA Form I was also collectedusing a X'PERT Pro Phillips X-ray diffractometer with a copper Kαradiation (wavelength 1.542 Å). The prominent 2θ positions along withthe d-spacings are summarized in Table 3A.

TABLE 3A SAHA Form I Position [° 2θ] d-spacing [Å] 9.1 9.7 10.8 8.2 12.37.2 17.2 5.2 19.2 4.6 19.8 4.5 23.7 3.7 24.1 3.7 25.7 3.5 26.8 3.3 27.73.2

Example 8 Melting Point Analysis

Melting point analysis was performed on SARA Form I-V.

TABLE 8 Melting Points SAHA Sample MP (° C.) SAHA Form I 159-160 SAHAForm II 152-155 SAHA Form III 138-144 SAHA Form IV  158-160.5 SAHA FormV 159.5-160.5

Example 9 Differential Scanning Calorimetric Analysis

Differential Scanning Calorimetric (DSC) analysis was performed on SAHAForm I-V.

Equipment:

Standard Aluminum DSC sample pans and covers used were Perkin Elmer(Part #0219-0041, or equivalent).Sample Pan Crimper Accessory used was a Perkin Elmer Standard AluminumPan Crimper or equivalent.Differential Scanning Calorimeter used was Perkin Elmer DSC 6 orequivalent.Micro Balance used was Perkin Elmer AD-4 Autobalance or equivalent.Software—Pyris or other suitable thermal analysis software.

Differential Scanning Calorimeter Conditions:

Purge Gas Nitrogen (about 20 mL/min)Cooling Agent Tap waterOven Temp Program Heat from 50° C. at 10.0° C./minute to at least 30° C.above the observed melting temperature.

Data Interpretation:

The peak temperature and melting onset temperatures were determined.Peak shapes were observed for any indication that more than one meltingtemperature is occurring.The results of multiple samples are summarized in Table 9:

TABLE 9 Differential Scanning Calorimetry SAHA Sample Onset Temp (° C.)Peak Temp (° C.) SAHA Form I 161.8 164.8 162.1 164.5 162.7 165.0 161.4164.7 161.9 164.1 161.6 164.3 152.5 164.9 160.9 163.7 161.5 163.5 161.58163.93 SAHA Form II 156.6 160.2 158.22, 161.58 (doublet) 160.39, 162.4(doublet) SAHA Form III 110.86, 145.68 (doublet) 120.11, 147.58(doublet) 114.69, 144.41 (doublet) 122.40, 147.00 (doublet) 123.67,148.89 (doublet) 127.89, 152.22 (doublet) SAHA Form IV 156.26, 161.64(doublet) 160.55, 153.66 (doublet) 160.46, 164.77 (doublet) 162.63,166.55 (doublet) SAHA Form V 124.47, 162.55 (doublet) 128.13, 165.14(doublet)

Depending upon the rate of heating, i.e. the scan rate, at which the DSCanalysis is conducted, the calibration standard used, instrumentcalibration, the relative humidity and upon the chemical purity, theendotherms of the respective SAHA analyzed may vary. For any givensample, the observed endotherm may also differ from instrument toinstrument; however it will generally be within the ranges definedherein provided the instruments are calibrated similarly.

Example 10 Development of Computer Simulation Model Model DevelopmentProcedure

During encapsulation, the SAHA crystals undergo breakage from thepressure of the tamping pins. The first part of the SAHA dissolution andbreakage modeling was the development of dissolution and breakagemodels. Both models were combined for the calculation of the breakageand subsequent dissolution of the broken crystals, and evaluation andoptimization of the model parameters for different batches.

The development procedure can be summarized as follows. First, particlesize distributions (PSD) and dissolution profiles of the SAHA Form Icrystals before encapsulation were measured. The model for dissolutionof poly-disperse powders was developed by combining the resistance ofintrinsic dissolution [32, 33] and the film resistance [34] forpolydisperse powders and crystals. The dissolution model parametersinclude the intrinsic dissolution constant and the shape factors ofnon-spherical crystals. The parameters of the dissolution model wereevaluated by fitting the model solutions to the experimental dissolutionprofile of the SAHA Form I crystals.

Encapsulation of the SAHA crystals with excipients was performed. Thedensity of the capsule content was evaluated for each experimentalcondition. The dissolution profile of SAHA from capsules was measured.Acceleration of the dissolution was observed as compared to thedissolution of the SAHA crystals before encapsulation. The accelerationof dissolution confirms breakage of the crystals during encapsulation.

The breakage model of the SARA crystals during encapsulation wasdeveloped [35, 36]. The breakage model parameters include the breakagerate constant and the breakage rate exponent. The breakage model wasemployed for calculation of the PSD after breakage during encapsulationassuming a combination of breakage rate constant and breakage rateexponent. The dissolution model was employed for calculation of thedissolution profile of the broken crystals with the calculated PSD. Thecomputed SAHA dissolution profile was compared with the experimentalSAHA dissolution profile for the capsule. The procedure in thisparagraph was repeated for different combinations of the breakage rateconstants and exponents until an optimum fit was found. The procedurewas also repeated for different batches of the SAHA crystals havingdifferent PSDs and for different encapsulation conditions.

The optimum dissolution model parameters were found which couldsatisfactorily describe dissolution of all batches having differentPSDs. The parameters were used for prediction of dissolution of both theSAHA crystals before and after encapsulation. The optimum breakage rateexponent was found and could be used in the breakage model for allbatches.

The optimum breakage rate constant for each batch and each encapsulationconditions was related to the capsule density at given encapsulationconditions. A near linear dependence was found between the increasingcapsule density and the breakage rate constant for all relevantencapsulation conditions and batches as illustrated in FIG. 11.

Prediction Procedure

After the breakage and dissolution model development and the modelparameter optimization, the combined breakage and dissolution modelcould be used for prediction of the dissolution profiles for new SAHAbatches and for the optimization of the encapsulation conditions. Theonly information needed is the PSD of the new batch.

The prediction procedure can be summarized as follows. First, the PSD ofa new batch before encapsulation was measured and capsule density wasassumed. The correlation between capsule density and the breakage rateconstant was used for calculation of a breakage rate constant.

The computed breakage rate constant together with the optimum breakagerate exponent were introduced into the breakage model to simulate thebreakage during encapsulation and a new PSD of broken crystals afterencapsulation was computed.

The new PSD was introduced into the dissolution model and the SAHAdissolution profile from a capsule was simulated. The simulateddissolution profile was compared with the target reference profile. Theprocedure was repeated for a new capsule density until the optimum fitbetween the simulated profiles and target was found. The optimum capsuledensity directly determines the optimum encapsulation conditions.

Example 11 Blending of SAHA Crystals

The above prediction procedure can be used for determining the blendingratio of different crystallization batches to obtain a dissolutionprofile similar to that of the reference.

Optimization of the Blending Ratio of a Large Crystal Batch 283 with aWet-Milled Crystal Batch 288

Mathematical models were used for the prediction of the optimal blend ofthe larger crystal batch 283 with the wet-milled crystal batch 288.First, the goal was to find optimum blends for capsules prepared atgiven conditions (capsule density) and then to find the most robustblend for different encapsulation conditions. An example of theoptimization is shown in FIG. 8 for the capsule density=0.8. Thedependence of the predicted F2 values for different capsule densitiesare shown in FIG. 9. FIG. 9 shows that the predicted F2 test valueincreases with the decrease of the capsule density (The decrease incapsule density causes a decrease in the extent of breakage). Thewet-milled crystals showed little breakage during the encapsulationprocess. It was concluded that the most robust blend composition (thelowest F2 variation between capsules prepared at different conditions)contained 30% of the batch 288 crystals and 70% of the batch 283crystals.

Experimental dissolution curves for capsules manufactured from the blendcontaining 30% of the batch 288 crystals and 70% of the batch 283crystals are presented in FIG. 10.

Blending of Crystallization Batches

A similar computer simulation process can be used for blending SAHAcrystals from different crystallization batches. Depending on theparticle sizes of the different batch crystals, one would take intoaccount the breakage constant of each batch. Using the computersimulation process above, capsule lot 0683_(—)007A001 was produced byblending 21.2% of batch 1002DRW, 18.0% of batch 1008D, 34.4% of batch1002E, 10.0% of batch 1004E and 16.4% of batch 1006D.

Batch 1001E and batch 1003E SAHA Polymorph I crystals were blendedwithout the aid of computer simulation, and were blended at a ratio of2:1 to produce capsule lot 6001.004.

Example 12 Powder Blending of SAHA Crystals Powder Blending

25.0 Kg of blended SAHA Polymorph I crystals were first sieved through a30 mesh screen (600 μm). The resulting SAHA, 11.1 Kg of MicrocrystallineCellulose (Avicel PH-101), and 1.13 Kg of Croscarmellose Sodium werethen loaded into the 141.6 L V-blender, 113 L Tote blender or anothercomparable sized and type blender. For the V-blender, the resultingmaterial was mixed to homogeneity for approximately 8 minutes atapproximately 25 rpm. For the Tote blender, the resulting material wasmixed to homogeneity for approximately 17 minutes at approximately 12rpm.

Powder Blend Lubrication

293.0 g of Magnesium Stearate (vegetable grade) was sieved through a 30mesh screen (600 μm) and loaded into the V-blender with the blendedpowder mixture. The resulting mixture was blended to homogeneity forapproximately 8 minutes at approximately 25 rpm. 293.0 g of MagnesiumStearate (vegetable grade) was also sieved through a 60 mesh screen (250μm) and loaded into a tote blender with the blended powder mixture. Theresulting mixture was blended to homogeneity for approximately 17minutes at approximately 12 rpm.

Table 10 summarizes the physical properties of the raw materials in thecapsule.

TABLE 10 Physical and Chemical Properties of Raw Materials. Raw MaterialPhysical Property Value Suberoylanilide hydroxamic Melting Point (DSC)161-163° C. acid (SAHA) - milled and Solubility: large crystals In Water<0.1 mg/ml In Methanol 42 mg/ml In Ethanol 0.1 mg/ml 2% CIP100 aqueoussoln. 11.3 mg/ml 2% SD-20 aqueous soln. 0.085 mg/ml MicrocrystallineCellulose Nominal Mean Particle Size 50 μm (Avicel PH-101) NF, Ph. Eur.,Moisture Content ≦5% JP (FMC BioPolymer) Bulk Density 0.26-0.31 g/ccCroscarmellose Sodium NF, Bulk Density 0.48 g/cc Ph. Eur., JP TappedDensity 0.67 g/cc (FMC BioPolymer) Particle Size Distribution ≦2% wt.retained on Mesh No. 200 (75 μm) ≦10% wt. retained on Mesh No. 325 (45μm) Magnesium Stearate Bulk Density 0.16 g/cc (vegetable grade) NF, Ph.Particle Size Distribution ≦2% wt. retained on Eur., JP Mesh No. 200 (75μm) (Mallinckrodt Baker Inc.) Specific Surface Area 4.2 ± 0.04 m²/g

Example 13 Encapsulation of SAHA Capsules Encapsulation/Weight Sorting

The lubed powder mixture was encapsulated using an H&K encapsulator,polished tamping pins or chromium nitride coated tamping pins and size“3” capsules to the desired capsule weight. The filled capsules werepolished using a capsule polisher and subsequently weight sorted using aweight sorter to the appropriate weight limit range. Table 11 summarizesthe encapsulator settings.

TABLE 11 Summary of Encapsulator Operational Settings Dosing Disc10.0-12.7 mm Tamping Pins/Station 3 or 12 Tamping Pin Type PolishedUncoated or chromium nitride coated Vacuum ON Encapsulator Speed 150-270caps/min or 750-1000 caps/min

The final SAHA Capsule Composition is illustrated in Table 12. Thecapsules are weight-sorted using an acceptance limit for capsule weightvariation of ±10% the target capsule weight. The capsule weightvariation in a typical batch is ±4% of the target capsule weight.

TABLE 12 SAHA Capsule Composition Ingredient Unit Weight (mg) Weight (%)Suberoylanilide X Y hydroxamic acid (SAHA) - Milled Suberoylanilide100.0 − x 66.67 − y hydroxamic acid (SAHA) - Large Microcrystalline44.33 29.80 Cellulose (Avicel PH- 101) NF, Ph. Eur., JP CroscarmelloseSodium 4.500 3.00 NF, Ph. Eur., JP Magnesium Stearate 1.170 0.78(vegetable grade) NF, Ph. Eur., JP Hard Gelatin Capsule, 49.00 N/A Size“3” Conisnap, White Opaque/White Opaque* Total** 150.0 100.00 *Themarket capsule ink formulation is Colorcon S-1-17762. TSE-free gelatincapsules. **Total weights do not include the hard gelatin capsuleshells.

Example 14 Measurement of Dissolution Rate of SAHA Capsules

The dissolution rate of SAHA from hard gelatin capsules was evaluatedusing a USP Dissolution Apparatus II (VK 7000, Varian Inc., Cary, N.C.).Each capsule was placed into a helical sinker (Quality Lab AccessoriesL.L.C., Manville, N.J.) and delivered to vessels containing 900 mL of2.0% Tween (TCI America, Portland, Oregon) at a temperature of 37±0.5°C. The paddles were rotated at 100 rpm and samples were pulled atspecified time intervals via an autosampler (VK 8000, Varian Inc., Cary,N.C.) equipped with 35 μm full flow filters (Varian Inc., Cary, N.C.).

Subsequently, samples were assayed for SAHA by High Performance LiquidChromatography (Agilent 1100 series, Agilent Technologies Inc.,Wilmington, Del.). The chromatographic analysis was conducted using aPhenomenex Luna C8 (2) (100×4.6 mm) 5 μm particle size column, a mobilephase of 1:1 methanol/0.1% trifluroacetic acid (Reagent Grade, Fisher),and a detection wavelength of 242 nm.

Excipients, capsule shell and moisture showed little effect on thedissolution rate of the SAHA capsule contents. However, the particlesize distribution of SAHA influenced the dissolution rate.

The dissolution rate profiles of SAHA from the capsule contents areillustrated in Tables 13, 14 and FIG. 5. The dissolution rate profile ofSAHA from the reference capsule Lot 0683_(—)004A001 is illustrated inFIG. 1. The F2 factors of SAHA from various capsule batches werecalculated using capsule Lot 0683_(—)004A001 as the reference.

TABLE 13 Dissolution Rate of SAHA Capsules Average SAHA % (with RMSD)Dissolved at Time (minutes) Capsule Lot # 0 10 15 20 30 45 60 F2 FactorC04-0306-001 0.0 58.5 68.9 75.0 82.9 89.4 93.1 58.2 0.0 6.5 5.2 4.4 3.01.8 1.1 F-613-001 0.0 63.3 72.7 78.5 85.7 91.9 95.4 49.7 0.0 2.8 2.3 1.51.1 1.3 1.0 6001.001 0.0 55.1 64.6 70.5 78.7 85.5 89.7 76.2 0.0 3.2 3.02.6 2.3 2.8 2.8 6001.002 0.0 43.9 52.9 59.0 67.7 76.1 81.7 55.3 0.0 8.77.1 6.4 4.8 3.6 3.1 6001.003 0.0 46.0 54.4 60.1 68.5 76.9 81.8 58.7 0.03.1 2.7 2.8 3.0 3.0 3.4 6001.004 0.0 51.2 60.0 66.1 74.5 82.5 87.9 89.70.0 2.8 2.3 1.9 2.3 2.4 2.1 6001.005 0.0 53.5 61.0 66.5 73.1 79.3 83.680.3 0.0 2.5 1.5 1.8 2.5 3.0 2.7 6001.006 0.0 39.2 49.4 55.8 61.0 68.873.9 43.9 0.0 0.4 6.3 8.7 3.3 4.1 4.2 0683_004A001 0.0 52.7 61.7 67.775.5 82.6 87.0 Reference Lot

TABLE 14 Dissolution Rate Profile of 0683_DFC007A001 Average SAHA %Dissolved at Time, minutes Capsule Density 0 10 15 20 30 45 60 F2 Factor0683_007A001 0.0 47 56 62 70 78 82 61.9 0.0 2.6 2.8 2.8 3.0 3.6 3.30683_004A001 0.0 52.7 61.7 67.7 75.5 82.6 87.0 Reference Lot 0.0 5.7 5.25.0 4.7 4.5 4.3

Example 15 Measurement of Dissolution Rate of SAHA API

The dissolution rate of 100 mg of SAHA API before encapsulation wasevaluated using a USP Dissolution Apparatus II (VK 7000, Varian Inc.,Cary, N.C.). About 100 mg of SAHA was delivered to vessels containing900 mL of 2.0% Tween (TCI America, Portland, Oreg.) at a temperature of37±0.5° C. The paddles were rotated at 100 rpm and samples were pulledat specified time intervals via an autosampler (VK 8000, Varian Inc.,Cary, N.C.) equipped with 35 μm full flow filters (Varian Inc., Cary,N.C.).

Subsequently, samples were assayed for SAHA by High Performance LiquidChromatography (Agilent 1100 series, Agilent Technologies Inc.,Wilmington, Del.). The chromatographic analysis was conducted using aPhenomenex Lima C8 (2) (100×4.6 mm) 5 μm particle size column, a mobilephase of 1:1 methanol/0.1% trifluroacetic acid (Reagent Grade, Fisher),and a detection wavelength of 242 nm.

The dissolution rate profiles of the SAHA API batches are illustrated inTable 15 and FIG. 6.

TABLE 15 Dissolution Rate Profiles of SAHA API batches Average SAHA %Dissolved at Time, minutes API Lot # 0 10 15 20 30 45 601136-1136-00-001 0.0 38.2 48.9 57.3 69.3 80.6 87.7 0.0 1.7 2.4 2.7 2.83.6 2.6 1376-C-RO-02 0.0 31.1 39.7 47.1 58.3 70.1 78.3 0.0 5.2 4.5 3.13.1 2.7 2.9 1008D 0.0 26.8 34.8 41.5 51.3 61.8 69.8 0.0 2.6 3.1 2.6 2.22.4 2.1 1007D 0.0 37.8 46.1 52.1 61.9 71.8 79.3 0.0 3.3 3.2 2.6 2.4 2.42.0

Example 16 Measurement of Particle Size Distribution

Particle size measurements of the blended SAHA crystals (ActivePharmaceutical Ingredient: API), lubricated formulation blend, andcapsule contents were determined via a Sympatec laser diffractionanalyzer (HELOS H1006, Clausthal-Zellerfeld, Germany) equipped with aRODOS powder dispersion system.

Approximately 150 mg of sample was manually delivered to the system andatomized through a laser beam using 0.1 bar air pressure. Data wascollected using a focal length lens of 850 or 1750-μm and the targetedobscuration range was 5-20%. The fraunhofer optical model was utilizedto deconvolute the sample scattering patterns to yield the resultantparticle size distributions.

The particle size distribution of the SAHA capsule contents areillustrated in Table 16 and FIG. 3. The particle size distribution ofthe SAHA API batches prior to encapsulation are illustrated in Table 17and FIG. 4. The particle size distribution of SAHA capsules preparedusing a Blend of API 288 (30% wet-milled) and 283 (70% Large crystals)are illustrated in Table 18. The normalized particle size distributionof SAHA from capsules prepared using different blends of wet-milled andlarge crystals are illustrated in Table 19 and FIG. 12. The particlesize distribution of Lot C0666001 SAHA capsules prepared using 30%wet-milled and 70% large crystals are illustrated in Table 20 and FIG.13. The particle size distribution of Lot C0667001 SAHA capsulesprepared using 30% wet-milled and 70% large crystals are illustrated inTable 21 and FIG. 14.

TABLE 16 Particle Size Distribution of SAHA Capsule Content % VolumeCapsule Lot # Particle C04- Size 0306- F- 0683 (μm) 001 613.001 6001.0016001.002 6001.003 6001.004 6001.005 6001.006 DFC004A001 9 5.892 6.8608.426 9.278 7.468 8.043 11.36 5.039 9.193 11 1.742 2.068 2.075 2.0961.776 1.933 2.516 1.421 2.140 13 1.844 2.214 2.104 2.060 1.783 1.9332.440 1.479 2.130 15 1.926 2.332 2.123 2.021 1.785 1.926 2.361 1.5262.113 18 3.004 3.636 3.180 2.955 2.669 2.866 3.399 2.351 3.125 22 4.1104.934 4.180 3.793 3.518 3.748 4.282 3.193 4.060 26 4.126 4.884 4.0613.623 3.443 3.636 4.009 3.204 3.918 31 5.082 5.892 4.853 4.288 4.1664.362 4.647 3.957 4.670 37 5.858 6.582 5.428 4.786 4.751 4.928 5.0824.600 5.245 43 5.496 5.952 4.969 4.404 4.442 4.573 4.587 4.370 4.833 505.892 6.144 5.229 4.671 4.772 4.880 4.792 4.753 5.130 60 7.444 7.4346.501 5.863 6.056 6.157 5.935 6.121 6.430 75 9.194 8.754 7.950 7.2207.540 7.623 7.228 7.777 7.903 90 7.188 6.594 6.220 5.678 5.977 6.0145.623 6.290 6.198 105 5.582 4.994 4.876 4.474 4.736 4.747 4.376 5.0574.873 125 5.524 4.836 4.920 4.560 4.846 4.839 4.372 5.233 4.943 1504.774 4.092 4.426 4.205 4.494 4.451 3.904 4.860 4.488 180 3.872 3.2603.804 3.810 4.116 4.022 3.404 4.400 3.910 210 2.658 2.196 2.776 3.0253.338 3.189 2.605 3.510 2.913 250 2.386 1.930 2.649 3.238 3.692 3.4182.691 3.840 2.873 300 1.922 1.520 2.266 3.176 3.778 3.350 2.570 3.9632.583 360 1.484 1.162 1.891 2.910 3.548 3.025 2.326 3.930 2.198 4301.172 0.876 1.623 2.536 2.951 2.471 1.988 3.623 1.778 510 0.762 0.5761.391 2.100 2.088 1.777 1.572 2.839 1.273 610 0.614 0.232 1.194 1.7291.351 1.210 1.086 1.843 0.768 730 0.326 0.048 0.691 1.093 0.644 0.7010.602 0.634 0.285 870 0.126 0.000 0.191 0.409 0.234 0.174 0.245 0.1270.030 1030 0.000 0.000 0.009 0.000 0.039 0.000 0.000 0.064 0.000 12300.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1470 0.000 0.0000.000 0.000 0.000 0.000 0.000 0.000 0.000 1750 0.000 0.000 0.000 0.0000.000 0.000 0.000 0.000 0.000

TABLE 17 Particle Size Distribution of SAHA API Batches % VolumeParticle Size API Lot # (μm) 1136-1136-00-001 1376-C-RO-02 1008D 1007D 91.77 1.76 2.02 2.92 11 0.52 0.45 0.45 0.87 13 0.55 0.46 0.44 0.91 150.58 0.47 0.44 0.95 18 0.91 0.72 0.66 1.48 22 1.27 0.99 0.88 2.03 261.33 1.00 0.89 2.06 31 1.72 1.27 1.14 2.58 37 2.14 1.55 1.42 3.05 432.22 1.56 1.49 2.95 50 2.68 1.83 1.83 3.28 60 4.00 2.60 2.76 4.39 756.30 3.81 4.35 5.96 90 6.44 3.63 4.44 5.29 105 6.36 3.42 4.42 4.72 1258.05 4.35 5.71 5.58 150 9.07 5.47 6.72 6.13 180 9.36 6.92 7.48 6.52 2107.73 7.14 6.88 5.78 250 7.96 9.27 8.24 6.63 300 6.82 10.42 8.86 6.70 3604.99 10.36 8.75 6.09 430 3.24 8.97 7.90 5.03 510 1.95 6.34 6.16 3.71 6101.21 3.60 4.07 2.54 730 0.66 1.40 1.48 1.26 870 0.24 0.23 0.11 0.45 10300.00 0.00 0.00 0.10 1230 0.00 0.00 0.00 0.00 1470 0.00 0.00 0.00 0.001750 0.00 0.00 0.00 0.00

TABLE 18 Particle Size Distribution of SAHA Capsules prepared using aBlend of API 288 (30% wet-milled) and 283 (70% Large) Par- ticle %Volume Size, Capsule Density μm 0.73 0.81 0.84 0.90 Biobatch 4.5 5.325.97 6.27 7.22 5.86 5.5 1.20 1.34 1.43 1.62 1.29 6.5 1.16 1.28 1.37 1.541.22 7.5 1.12 1.23 1.32 1.47 1.17 9 1.59 1.75 1.89 2.09 1.68 11 2.002.21 2.39 2.61 2.15 13 1.88 2.08 2.26 2.45 2.11 15.5 2.22 2.48 2.68 2.882.64 18.5 2.51 2.83 3.06 3.25 3.21 21.5 2.39 2.72 2.92 3.09 3.25 25 2.693.08 3.28 3.44 3.8 30 3.69 4.25 4.49 4.67 5.32 37.5 5.28 6.07 6.35 6.537.45 45 4.99 5.68 5.88 5.97 6.63 52.5 4.66 5.27 5.39 5.42 5.81 62.5 5.676.34 6.42 6.37 6.57 75 6.28 6.89 6.88 6.75 6.66 90 6.63 7.06 6.95 6.726.34 105 5.89 6.02 5.81 5.53 5.02 125 6.92 6.69 6.28 5.84 5.15 150 7.316.52 5.87 5.29 4.7 180 6.83 5.51 4.69 4.09 3.98 215 5.43 3.85 3.13 2.673.19 255 3.53 2.04 1.75 1.49 2.36 305 1.95 0.71 0.95 0.81 1.61 365 0.780.12 0.20 0.13 0.84 435 0.06 0.00 0.06 0.06 0 515 0.00 0.00 0.00 0.00 0615 0.00 0.00 0.00 0.00 0 735 0.00 0.00 0.00 0.00 0 875 0.00 0.00 0.000.00 0

TABLE 19 Normalized Particle Size Distribution of SAHA from capsulesprepared using different blends of wet-milled and large crystals %Volume SAHA 100 mg 5W3/4U1; 5W3/4U1; 5W3/4U1; 5W3/4U1; 5W5/4U3; 5W5/4U3;5W5/4U3; 5W5/4U3; 5W5/4U3; 5W11/4U4; 5W12/4U4; 60:40; 60:40; 60:40;70:30; 70:30; 70:30; 80:20; 90:10; 90:10; 70:30; 70:30; Particle f2 = 78f2 = 64 f2 = 59 f2 = 79 f2 = 72 f2 = 41 f2 = 50 f2 = 48 f2 = 44 f2 = 46f2 = 48 Size ρ = ρ = ρ = ρ = ρ = ρ = ρ = ρ = ρ = ρ = ρ = (μm) 0.75 g/mL0.79 g/mL 0.82 g/mL 0.76 g/mL 0.69 g/mL 0.82 g/mL 0.80 g/mL 0.68 g/mL0.81 g/mL 0.69 g/mL 0.70 g/mL 5 9.850 11.86 11.95 9.352 4.525 7.4027.028 4.052 7.268 5.281 5.442 6 2.103 2.486 2.488 1.953 1.080 1.6731.553 0.857 1.554 1.168 1.227 7 1.967 2.306 2.322 1.802 1.062 1.6251.491 0.810 1.447 1.106 1.180 8 1.831 2.125 2.156 1.681 1.044 1.5631.429 0.763 1.371 1.059 1.133 9 2.513 2.910 2.942 2.303 1.519 2.2452.052 1.074 1.904 1.504 1.607 11 2.983 3.425 3.471 2.729 1.945 2.8052.551 1.322 2.330 1.841 1.988 13 2.630 3.028 3.045 2.406 1.859 2.6152.377 1.222 2.155 1.681 1.828 16 2.885 3.313 3.315 2.661 2.188 3.0332.750 1.417 2.500 1.877 2.054 19 2.988 3.460 3.417 2.808 2.454 3.3293.046 1.594 2.780 1.994 2.186 22 2.616 3.073 2.971 2.509 2.304 3.0602.821 1.533 2.630 1.770 1.962 25 2.741 3.243 3.096 2.680 2.578 3.3343.110 1.763 2.934 1.896 2.088 30 3.540 4.203 3.939 3.537 3.539 4.4294.190 2.502 4.029 2.516 2.767 38 4.831 5.671 5.274 4.901 5.186 6.1505.940 3.823 5.735 3.600 3.939 45 4.361 5.024 4.671 4.461 5.013 5.6215.514 3.902 5.413 3.441 3.721 53 3.818 4.333 4.054 3.962 4.677 5.0485.046 3.937 5.048 3.120 3.386 63 4.277 4.792 4.542 4.540 5.611 5.8786.038 5.271 6.204 3.520 3.874 75 4.358 4.813 4.653 4.798 6.270 6.3156.712 6.791 7.086 3.467 3.940 90 4.504 4.781 4.725 5.107 7.068 6.6097.228 8.509 7.751 3.361 4.042 105 4.275 4.197 4.274 4.894 6.870 5.8176.407 8.637 6.870 3.177 3.991 125 5.623 4.981 5.192 6.329 8.573 6.4827.132 10.88 7.535 4.599 5.738 150 6.677 5.219 5.608 7.338 8.886 5.9356.480 11.11 6.661 6.735 7.874 180 6.733 4.637 5.129 7.201 7.295 4.3894.698 8.825 4.641 9.030 9.486 215 5.546 3.333 3.765 5.630 4.625 2.6092.697 5.339 2.535 10.29 9.470 255 3.640 1.829 2.067 3.192 2.320 1.2821.266 2.528 1.178 9.571 7.595 305 1.934 0.747 0.777 1.117 1.029 0.5550.599 1.044 0.599 7.539 5.120 365 0.629 0.243 0.184 0.139 0.392 0.228−0.127 0.377 −0.127 3.802 2.392 435 0.149 −0.029 −0.029 −0.029 0.090−0.029 −0.029 0.119 −0.029 1.053 −0.029 515 0.000 0.000 0.000 0.0000.000 0.000 0.000 0.000 0.000 0.000 0.000 615 0.000 0.000 0.000 0.0000.000 0.000 0.000 0.000 0.000 0.000 0.000 735 0.000 0.000 0.000 0.0000.000 0.000 0.000 0.000 0.000 0.000 0.000 875 0.000 0.000 0.000 0.0000.000 0.000 0.000 0.000 0.000 0.000 0.000 Large 141 141 141 141 122 122122 122 122 182 165 D_(mean) (μm) Milled 29 29 29 29 34 34 34 34 34 3636 D_(mean) (μm)

TABLE 20 Particle Size Distribution of Lot C0666001 SAHA capsulesprepared using 30% wet- milled and 70% large crystals % Volume SAHA 100mg Lot # C0666001 Capsule, Capsule, Capsule, Capsule, Capsule, Capsule,Capsule, Particle Capsule, Drum Drum Drum Drum Drum Drum Drum Size Drum1B, 2B, 3A, 3B, 4A, 4B, 5B, (μm) 1A, cap4 cap3 cap1 cap4 cap1 cap4 cap4cap3 5 5.91 6.03 5.57 5.48 5.56 5.49 5.81 5.99 6 1.31 1.33 1.22 1.201.23 1.18 1.25 1.30 7 1.25 1.26 1.16 1.14 1.17 1.12 1.18 1.23 8 1.201.21 1.10 1.09 1.13 1.06 1.12 1.17 9 1.71 1.72 1.56 1.55 1.61 1.50 1.571.66 11 2.15 2.16 1.95 1.95 2.03 1.88 1.95 2.08 13 2.03 2.04 1.84 1.841.93 1.78 1.83 1.96 16 2.42 2.43 2.20 2.20 2.30 2.13 2.19 2.33 19 2.782.80 2.54 2.54 2.66 2.48 2.54 2.69 22 2.69 2.71 2.49 2.48 2.59 2.44 2.502.61 25 3.06 3.09 2.86 2.85 2.97 2.82 2.89 2.99 30 4.25 4.31 4.03 4.034.17 4.01 4.08 4.19 38 6.13 6.22 5.85 5.92 6.08 5.88 5.95 6.07 45 5.755.83 5.51 5.61 5.72 5.56 5.60 5.70 53 5.29 5.36 5.08 5.20 5.29 5.14 5.175.25 63 6.31 6.39 6.08 6.24 6.34 6.17 6.20 6.28 75 6.84 6.92 6.62 6.836.92 6.75 6.77 6.84 90 7.10 7.13 6.92 7.19 7.23 7.11 7.11 7.13 105 6.166.14 6.07 6.36 6.32 6.30 6.28 6.20 125 6.89 6.80 6.88 7.30 7.13 7.267.17 6.95 150 6.59 6.44 6.71 7.22 6.90 7.23 7.03 6.65 180 5.32 5.15 5.526.02 5.66 6.10 5.81 5.36 215 3.56 3.42 3.79 4.13 3.85 4.28 3.98 3.60 2551.97 1.87 2.21 2.34 2.10 2.48 2.27 2.03 305 0.96 0.89 1.25 1.28 0.891.28 1.19 1.07 365 0.38 0.34 0.72 0.00 0.23 0.57 0.55 0.46 435 0.00 0.000.58 0.00 0.00 0.00 0.00 0.19 515 0.00 0.00 0.63 0.00 0.00 0.00 0.000.00 615 0.00 0.00 0.66 0.00 0.00 0.00 0.00 0.00 735 0.00 0.00 0.39 0.000.00 0.00 0.00 0.00 875 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

TABLE 21 Particle Size Distribution of Lot C0667001 SAHA capsulesprepared using 30% wet- milled and 70% large crystals % Volume SAHA 100mg Lot # C0667001 Particle capsule capsule capsule capsule capsulecapsule capsule capsule Size (μm) L10C4 L1C4 L1C3 L2C3 L9C4 L5C3 L10C2L5C1 5 5.57 5.46 5.08 4.86 4.44 4.07 3.78 4.09 6 1.21 1.20 1.12 1.040.97 0.90 0.86 0.91 7 1.15 1.14 1.06 0.98 0.92 0.86 0.83 0.87 8 1.101.09 1.01 0.93 0.87 0.83 0.80 0.83 9 1.57 1.55 1.44 1.32 1.25 1.19 1.171.20 11 1.98 1.96 1.83 1.66 1.58 1.53 1.52 1.53 13 1.91 1.89 1.76 1.611.54 1.50 1.49 1.50 16 2.33 2.31 2.17 2.00 1.92 1.87 1.87 1.86 19 2.772.74 2.59 2.43 2.33 2.27 2.27 2.26 22 2.77 2.75 2.62 2.49 2.39 2.33 2.322.31 25 3.24 3.20 3.08 2.98 2.86 2.78 2.76 2.75 30 4.62 4.54 4.40 4.324.16 4.05 4.00 3.99 38 6.76 6.58 6.44 6.44 6.21 6.07 5.99 5.97 45 6.356.16 6.07 6.16 5.97 5.86 5.76 5.77 53 5.86 5.67 5.62 5.77 5.62 5.54 5.435.44 63 7.03 6.79 6.75 7.03 6.88 6.82 6.69 6.68 75 7.68 7.41 7.41 7.797.72 7.69 7.58 7.51 90 7.93 7.66 7.73 8.17 8.27 8.28 8.22 8.07 105 6.726.51 6.63 7.02 7.31 7.36 7.37 7.16 125 7.17 7.00 7.18 7.62 8.19 8.298.38 8.05 150 6.29 6.24 6.42 6.87 7.58 7.79 7.96 7.57 180 4.40 4.57 4.645.07 5.61 5.96 6.19 5.85 215 2.35 2.77 2.74 3.05 3.24 3.61 3.87 3.73 2550.92 1.47 1.45 1.51 1.48 1.72 1.92 2.09 305 0.32 0.83 0.88 0.65 0.680.66 0.76 1.19 365 0.00 0.51 0.67 0.19 0.00 0.19 0.22 0.60 435 0.00 0.000.67 0.05 0.00 0.00 0.00 0.24 515 0.00 0.00 0.53 0.00 0.00 0.00 0.000.00 615 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 735 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 875 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Example 17 Patient Studies

This Phase I study conducted in advanced stage cancer patients assessedsafety and tolerability of oral vorinostat administered 400 mg q.d.,single- and multiple-dose serum pharmacokinetics (PK) of vorinostat, andthe effect of a standard high-fat meal on single-dose vorinostat PK.Patients received a single-dose of 400 mg vorinostat on Day 1 (fasted)and Day 5 (after a standard high-fat meal) with 48 hours of postdose PKsampling on both days. Patients then received 400 mg vorinostat once aday on Days 7 through 28 (22 days of dosing). On Day 28, vorinostat wasadministered after a standard high-fat meal with PK sampling for 24 hrspostdose. Of 23 patients enrolled, 23 were evaluable for Day 1 PK, 20for Day 5 PK, and 14 for Day 28 PK. The apparent t_(1/2) of vorinostatwas short. A high-fat meal was associated with a small increase in theextent of absorption and a modest decrease in the rate of absorption ofvorinostat. A lag time of at least 15 minutes was observed beforedetectable levels of vorinostat were observed in serum in the fed statein most subjects, and T_(max) was delayed. Following multiple-doseadministration of vorinostat, serum concentration time profiles weresimilar to those of single-dose administration. Trough concentrationsfollowing multiple-dose administration were generally below the limit ofquantification, which is consistent with the short apparent terminalt_(1/2). In conclusion, short-term administration of vorinostat topatients with advanced cancer was generally well tolerated. Vorinostatexhibited a short t_(1/2), serum concentration time profiles that weresimilar between single-dose and multiple-dose administration, and aslightly decreased rate of absorption when administrated with a high-fatmeal.

TABLE 22 PK Parameters of Vorinostat Following Single and Multiple Dosesof Vorinostat 400 mg Daily Multiple Dose Single Dose Single Dose Doses*GMR^(†) p-Value Diet Fasted Fed Fed — — N 23 20 14 — — AUC_(0-∞,) μM ·hr^(‡) 3.87 5.33 — 1.38^(§) <0.001^(§) (Range) (2.33-9.86) (3.41-9.34)(4.00-10.36) — — AUC_(0-24 hr), μM · hr^(‡) 3.82 5.33 6.46 1.21^(||);1.23^(¶) 0.019^(||); 0.010^(¶) C_(max), μM^(‡) 1.12 1.02 1.13 0.91^(§)0.451^(§) T_(max), hr^(#) 1.50 4.00 4.21 — <0.001^(§); 0.869** t_(1/2),hr^(††) 1.74 1.44 1.34 — 0.036^(§) f_(e),^(‡‡) 0.0021 0.0030 0.0037 — —fe = Fraction of dose excreted unchanged in urine. *Once daily for 22days. ^(†)Geometric mean ratio. ^(‡)Geometric mean. ^(§)Single dosefed/single dose fasted. ^(||)Accumulation ratio: AUC_(0-24 hr) multipledose fed/AUC_(0-24 hr) single dose fed. ^(¶)Linearity ratio:AUC_(0-24 hr) multiple dose fed/AUC_(0-∞) single dose fed. ^(#)Median.**Multiple dose fed/single dose fed. ^(††)Harmonic mean. ^(‡‡)Arithmeticmean (single dose fasted n = 22, single dose fed n = 21, multiple dosefed n = 12.

While this invention has been particularly shown and described withreferences to embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the meaning of the invention described.Rather, the scope of the invention is defined by the claims that follow:

REFERENCES

-   1. Sporn, M. B., Roberts, A. B., and Driscoll, J. S. (1985) in    Cancer: Principles and Practice of Oncology, eds. Hellman, S.,    Rosenberg, S. A., and DeVita, V. T., Jr., Ed. 2, (J. B. Lippincott,    Philadelphia), P. 49.-   2. Breitman, T. R., Selonick, S. E., and Collins, S. J. (1980) Proc.    Natl. Acad. Sci. USA 77: 2936-2940.-   3. Olsson, I. L. and Breitman, T. R. (1982) Cancer Res. 42:    3924-3927.-   4. Schwartz, E. L. and Sartorelli, A. C. (1982) Cancer Res. 42:    2651-2655.-   5. Marks, P. A., Sheffery, M., and Rifkind, R. A. (1987) Cancer Res.    47: 659.-   6. Sachs, L. (1978) Nature (Lond.) 274: 535.-   7. Friend, C., Scher, W., Holland, J. W., and Sato, T. (1971) Proc.    Natl. Acad. Sci. (USA) 68: 378-382.-   8. Tanaka, M., Levy, J., Terada, M., Breslow, R., Rifkind, R. A.,    and Marks, P. A. (1975) Proc. Natl. Acad. Sci. (USA) 72: 1003-1006.-   9. Reuben, R. C., Wife, R. L., Breslow, R., Rifkind, R. A., and    Marks, P. A. (1976) Proc. Natl. Acad. Sci. (USA) 73: 862-866.-   10. Abe, E., Miyaura, C., Sakagami, H., Takeda, M., Konno, K.,    Yamazaki, T., Yoshika, S., and Suda, T. (1981) Proc. Natl, Acad,    Sci. (USA) 78: 4990-4994.-   11. Schwartz, E. L., Snoddy, J. R., Kreutter, D., Rasmussen, H., and    Sartorelli, A. C. (1983) Proc. Am. Assoc. Cancer Res. 24: 18.-   12. Tanenaga, K., Hozumi, M., and Sakagami, Y. (1980) Cancer Res.    40: 914-919.-   13. Lotem, J. and Sachs, L. (1975) Int. J. Cancer 15: 731-740.-   14. Metcalf, D. (1985) Science, 229: 16-22.-   15. Scher, W., Scher, B. M., and Waxman, S. (1983) Exp. Hematol. 11:    490-498.-   16. Scher, W., Scher, B. M., and Waxman, S. (1982) Biochem. &    Biophys. Res. Comm 109: 348-354.-   17. Huberman, E. and Callaham, M. F. (1979) Proc. Natl. Acad. Sci.    (USA) 76: 1293-1297.-   18. Lottem, J. and Sachs, L. (1979) Proc. Natl. Acad. Sci. (USA) 76:    5158-5162.-   19. Terada, M., Epner, E., Nudel, U., Salmon, J., Fibach, E.,    Rifkind, R. A., and Marks, P. A. (1978) Proc. Natl. Acad. Sci. (USA)    75: 2795-2799.-   20. Morin, M. J. and Sartorelli, A. C. (1984) Cancer Res. 44:    2807-2812.-   21. Schwartz, E. L., Brown, B. J., Nierenberg, M., Marsh, J. C., and    Sartorelli, A. C. (1983) Cancer Res. 43: 2725-2730.-   22. Sugano, H., Furusawa, M., Kawaguchi, T., and Ikawa, Y. (1973)    Bibl. Hematol. 39: 943-954.-   23. Ebert, P. S., Wars, I., and Buell, D. N. (1976) Cancer Res. 36:    1809-1813.-   24. Hayashi, M., Okabe, J., and Hozumi, M. (1979) Gann 70: 235-238.-   25. Richon, V. M., Webb, Y., Merger, R., et al. (1996) PNAS    93:5705-8.-   26. Cohen, L. A., Amin, S., Marks, P. A., Rifkind, R. A., Desai, D.,    and Richon, V. M. (1999) Anticancer Research 19:4999-5006.-   27. Grunstein, M. (1997) Nature 389:349-52.-   28. Finnin, M. S., Donigian, J. R., Cohen, A., et al. (1999) Nature    401:188-193.-   29. Van Lint, C., Emiliani, S., Verdin, E. (1996) Gene Expression    5:245-53.-   30. Archer, S. Shufen, M. Shei, A., Hodin, R. (1998) PNAS    95:6791-96.-   31. Dressel, U., Renkawitz, R., Baniahmad, A. (2000) Anticancer    Research 20(2A):1017-22.-   32. Parker, Vigoroux, Reed, AIChE J. (2000) pp. 1290-99.-   33. Nunez, Espiell, Chem. Eng. Sci. (1986) pp. 2075-83.-   34. O. Levenspiel: Chemical Reaction Engineering, 2nd Ed., p. 373.-   35. M. Vanni: J. of Colloid and Interface Sci. (2000) pp. 143-160.-   36. P. J. Hill and K. M. Ng, AIChE J. (1995) pp. 1204-1216.

1-39. (canceled)
 40. A method of producing recrystallized activeingredient of suberoylanilide hydroxamic acid or a pharmaceuticallyacceptable salt or hydrate thereof, comprising the steps of: (a)providing crystalline active ingredient to an organic solvent, water ora mixture thereof to form a slurry; (b) heating the slurry to establish2-30% undissolved crystalline active ingredient; and (c) cooling theslurry to obtain the recrystallized active ingredient.
 41. The method ofclaim 40, wherein the crystalline active ingredient in step (a) has amean particle size less than about 60 μm.
 42. The method of claim 40,wherein the crystalline active ingredient is prepared by the steps of:(i) adding crystalline active ingredient to an organic solvent, water ormixture thereof to form a seed slurry; and (ii) wet-milling the slurryto achieve wet-milled crystalline active ingredient.
 43. The method ofclaim 40, wherein the crystalline active ingredient is prepared by thestep of dry-milling crystalline active ingredient.
 44. The method ofclaim 40, wherein the crystalline active ingredient is obtained in thepresence of hydroxylamine.
 45. The method of claim 40, wherein in step(a), a mixture of 40-99% ethanol and 60-1% water is used.
 46. (canceled)47. The method of claim 45, wherein in step (b), the slurry is heated to60-75° C. for about 1-3 hours.
 48. The method of claim 47, wherein step(c) is performed by cooling from between 60 to 75° C. to between 25 to−5° C. in about 15 to 72 hours.
 49. The method of claim 48, wherein theactive ingredient is suberoylanilide hydroxamic acid.
 50. A method ofproducing recrystallized active ingredient of suberoylanilide hydroxamicacid or a pharmaceutically acceptable salt or hydrate thereof,comprising the steps of: (a) providing crystalline active ingredient toan organic solvent, water or a mixture thereof to a first vessel to forma slurry; (b) heating the slurry in the first vessel to dissolvesubstantially all of the crystalline active ingredient; (c) cooling thecontents in step (b) in the first vessel to a temperature thatsupersaturates the solution. (d) adding seeds of the crystalline activeingredient to the contents of step (c); (e) aging the contents of step(d) at the same temperature as step (c); (f) cooling the contents instep (e) to obtain the recrystallized active ingredient.
 51. The methodof claim 50, wherein step (d) comprises the steps of: (i) providingcrystalline active ingredient in an organic solvent, water or mixturethereof to form a seed slurry; (ii) heating and aging the seed slurry todissolve a portion of the seeds; (iii) cooling the contents in step (ii)to the same temperature as in step (c); (iv) transferring the seedslurry in step (iii) to the first vessel.
 52. The method of claim 51,wherein the crystalline active ingredient of step (i) has a meanparticle size less than about 60 μm.
 53. The method of claim 51, whereinstep (i) is prepared by the steps of: (v) adding crystalline activeingredient to an organic solvent, water or mixture thereof to form aseed slurry; (vi) wet-milling the slurry to achieve wet-milledcrystalline active ingredient.
 54. The method of claim 51, wherein step(i) is prepared by the steps of: (v) dry-milling crystalline activeingredient; (vi) adding the dry-milled crystalline active ingredient toan organic solvent, water or mixture thereof to form a seed slurry. 55.The method of claim 53, wherein after step (vi), further comprising thestep of isolating, washing and drying the wet-milled crystalline activeingredient prior to step (d).
 56. The method of claim 50, wherein thecrystalline active ingredient of step (a) is obtained in the presence ofhydroxylamine.
 57. The method of claim 52, wherein a mixture of 40-99%ethanol and 60-1% water is used in step (a) and (i).
 58. The method ofclaim 52, wherein a mixture of ethanol to water ratio of 49:51 to 51:49is used in step (a) and (i)
 59. The method of claim 57, wherein in step(b), the slurry is heated to 60-75° C. under minimum of 15 psigpressure.
 60. The method of claim 58, wherein in step (b), the slurry isheated to 67-70° C. under minimum of 15 psig pressure.
 61. The method ofclaim 59, wherein in step (c), the contents are cooled to 60-65° C. 62.The method of claim 60, wherein in step (c), the contents are cooled to61-63° C.
 63. The method of claim 61, wherein in step (ii), the seedslurry is heated to 62-66° C.
 64. The method of claim 62, wherein instep (ii), the seed slurry is heated to 64-65° C.
 65. The method ofclaim 63, wherein step (f) is performed by cooling from between 60 to70° C., to between 25 to −5° C. in about 15 to 72 hours.
 66. The methodof claim 64, wherein step (f) is performed by cooling from between 60 to64° C., to between 0 to 10° C. in about 15 to 72 hours.
 67. The methodof claim 58, wherein the active ingredient is suberoylanilide hydroxamicacid. 68-74. (canceled)
 75. The method of claim 59, wherein the activeingredient is suberoylanilide hydroxamic acid.
 76. The method of claim60, wherein the active ingredient is suberoylanilide hydroxamic acid.77. The method of claim 61, wherein the active ingredient issuberoylanilide hydroxamic acid.
 78. The method of claim 62, wherein theactive ingredient is suberoylanilide hydroxamic acid.
 79. The method ofclaim 63, wherein the active ingredient is suberoylanilide hydroxamicacid.
 80. The method of claim 64, wherein the active ingredient issuberoylanilide hydroxamic acid.
 81. The method of claim 65, wherein theactive ingredient is suberoylanilide hydroxamic acid.
 82. The method ofclaim 66, wherein the active ingredient is suberoylanilide hydroxamicacid.